U.S. patent application number 14/381119 was filed with the patent office on 2015-10-08 for iaspp phosphorylation and metastatic potential.
The applicant listed for this patent is THE CHANCELLOR, MASTERS AND SCHOLARS OF THE UNIVERSITY OF OXFORD, LUDWIG INSTITUTE FOR CANCER RESEARCH LTD.. Invention is credited to Min Lu, Xin Lu.
Application Number | 20150285810 14/381119 |
Document ID | / |
Family ID | 47892029 |
Filed Date | 2015-10-08 |
United States Patent
Application |
20150285810 |
Kind Code |
A1 |
Lu; Xin ; et al. |
October 8, 2015 |
IASPP PHOSPHORYLATION AND METASTATIC POTENTIAL
Abstract
Described herein are methods of evaluating metastatic potential
of a cancer based on the phosphorylation state and/or intracellular
localization of iASPP. Therapeutic regimens designed based on the
results of the diagnostic methods are also provided.
Inventors: |
Lu; Xin; (London, GB)
; Lu; Min; (Oxford, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LUDWIG INSTITUTE FOR CANCER RESEARCH LTD.
THE CHANCELLOR, MASTERS AND SCHOLARS OF THE UNIVERSITY OF
OXFORD |
New York
Oxford |
NY |
US
GB |
|
|
Family ID: |
47892029 |
Appl. No.: |
14/381119 |
Filed: |
March 1, 2013 |
PCT Filed: |
March 1, 2013 |
PCT NO: |
PCT/US2013/028696 |
371 Date: |
August 26, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61711616 |
Oct 9, 2012 |
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61606090 |
Mar 2, 2012 |
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Current U.S.
Class: |
424/139.1 ;
435/7.1; 506/9; 514/19.3; 530/350; 530/387.9; 702/19 |
Current CPC
Class: |
C07K 16/30 20130101;
A61P 35/00 20180101; C07K 2317/34 20130101; G01N 2800/56 20130101;
G01N 2440/14 20130101; A61K 2039/505 20130101; G01N 2333/4704
20130101; C07K 2317/76 20130101; C07K 14/4703 20130101; G06F 19/00
20130101; G16H 50/30 20180101; C07K 16/18 20130101; C07K 16/44
20130101; C07K 14/4746 20130101; G01N 2333/4748 20130101; G01N
33/57496 20130101 |
International
Class: |
G01N 33/574 20060101
G01N033/574; C07K 14/47 20060101 C07K014/47; G06F 19/00 20060101
G06F019/00; C07K 16/18 20060101 C07K016/18 |
Claims
1. A method of evaluating metastatic potential of a cancer
comprising: (a) measuring phosphorylation state and/or
intracellular localization of inhibitory Apoptosis-Stimulating
Protein of p53 (iASPP) in a cancer cell from a mammalian subject,
and (b) diagnosing metastatic potential of the cancer based on the
phosphorylation state and/or intracellular localization of iASPP in
the cancer cell measured according to step (a).
2. The method of claim 1, comprising measuring phosphorylation
state and/or intracellular localization of iASPP in a plurality of
cancer cells from the mammalian subject, and diagnosing metastatic
potential of the cancer based on an average measurement from the
plurality of the cells, or from a percentage of the cells that
exceed a cutoff or reference measurement.
3. The method of claim 1, wherein the diagnosing of metastatic
potential is made from the percentage of total iASPP that is
phosphorylated or the percentage of total iASPP that is localized
in the nucleus of the cell.
4. The method of claim 1, wherein the mammalian subject is
human.
5. The method of claim 1, wherein the cancer cell is from a primary
tumor from the subject.
6. The method of claim 1, further comprising: screening the cancer
cell for at least one parameter selected from the group consisting
of: a p53 mutation that reduces a tumor suppressive function of
p53, an elevated measurement of mdm2 protein or mRNA, and a
B-RafV600E mutation; and diagnosing metastatic potential of the
cancer based on the phosphorylation state and/or intracellular
localization of iASPP in the cancer cell and from the presence or
absence of the at least one parameter.
7. (canceled)
8. The method of claim 4, wherein the measuring comprises measuring
the phosphorylation state of iASPP in the cancer cell, wherein the
phosphorylation is serine phosphorylation at an iASPP serine
residue corresponding to serine 84 (Ser84) and/or serine 113
(Ser113) in the human iASPP amino acid sequence of SEQ ID NO:
2.
9. The method of claim 8, wherein the measuring of the
phosphorylation state comprises at least one of: an analytical
technique that provides an absolute measure of phosphorylated
iASPP; an analytical technique that provides a relative measure of
phophorylated iASPP to total iASPP in the cell; and an analytical
technique that provides a relative measure of phosphorylated iASPP
to unphosphorylated iASPP in the cell.
10. The method of claim 1, that comprises contacting the cancer
cell, or protein isolated from the cancer cell, with a binding
partner that specifically binds phosphorylated iASPP, and measuring
the phosphorylation state of iASPP by measuring the amount of the
binding partner-iASPP complex formed.
11. The method of claim 10, wherein the binding partner comprises a
detectable label.
12. The method of claim 1 that comprises contacting the cancer
cell, or protein isolated from the cancer cell, with a first
binding partner and a second binding partner, wherein the first
binding partner binds iASPP and the second binding partner
specifically binds the phosphorylated iASPP, wherein the second
binding partner comprises a detectable label, and measuring the
phosphorylation state of iASPP by measuring the amount of second
binding partner-iASPP complex formed.
13. The method of claim 12, wherein the phosphorylated iASPP
recognized by the second binding partner comprises one or more
phosphorylated serine residues.
14. The method of claim 13, wherein the serine residues are
selected from the group consisting of Ser84 and Ser113.
15. The method of claim 12, wherein the first binding partner is an
antibody that binds to an epitope comprising all or some of amino
acids 501-510 of SEQ ID NO: 2 and the second binding partner is an
antibody that binds to an epitope comprising all or some of amino
acids 107-118 of SEQ ID NO: 2, wherein the amino acid at position
113 of SEQ ID NO: 2 is phosphorylated in the epitope of the second
binding partner.
16. The method of claim 1 that comprises contacting the cancer
cell, or protein isolated from the cancer cell, with a first
binding partner and a second binding partner that binds iASPP,
wherein the first binding partner binds phosphorylated and
unphosphorylated iASPP, and wherein the second binding partner
binds an epitope of iASPP that includes Ser84 or Ser113 of iASPP
(SEQ ID NO: 2) and selectively binds iASPP when said serine in said
epitope is unphosphorylated, and wherein the second binding partner
comprises a detectable label, and measuring the phosphorylation
state of iASPP by measuring the amount of second binding
partner-iASPP complex formed.
17. (canceled)
18. The method of claim 16, wherein the first binding partner is an
antibody that binds to an epitope comprising all or some of amino
acids 501-510 of SEQ ID NO: 2 and the second binding partner is an
antibody that binds to an epitope comprising all or some of amino
acids 111-122 of SEQ ID NO: 2.
19. The method of claim 1 that comprises measuring total iASPP in
the cell, and measuring unphosphorylated iASPP in the cell, wherein
a difference between total iASPP and unphosphorylated iASPP
provides an indication of the amount of phosphorylated iASPP in the
cell.
20. The method of claim 9, wherein measuring unphosphorylated iASPP
in the cell comprises contacting the cancer cell, or protein
isolated from the cancer cell, with a binding partner that
preferentially binds iASPP in which one or more serine residues
selected from serine 84 (Ser84, SEQ ID NO: 2) and serine 113 (Sen
13, SEQ ID NO: 2) is not phosphorylated.
21. The method of claim 9 that comprises contacting the cancer
cell, or protein isolated from the cancer cell, with a binding
partner that preferentially binds iASPP in which one or more serine
residues selected from serine 84 (Ser84, SEQ ID NO: 2) and serine
113 (Sen 13, SEQ ID NO: 2) is phosphorylated, and measuring the
phosphorylation state of iASPP by measuring the amount of
polypeptide-binding partner complex formed, to provide a measure of
iASPP that is phosphorylated at one or more of the serine
residues.
22. The method of claim 21, wherein the binding partner is an
antibody that preferentially recognizes an iASPP epitope in which
one or more serine residues selected from serine 84 (Ser84, SEQ ID
NO: 2) and serine 113 (Sen 13, SEQ ID NO: 2) is phosphorylated,
compared to epitopes of iASPP in which Ser84 and Sen 13 are
unphosphorylated.
23. The method of claim 21, wherein the antibody binds to an
epitope comprising all or some of amino acids 111-122 of SEQ ID NO:
2.
24. The method of any of the preceding claims, wherein the binding
is measured in situ in a tumor biopsy.
24. The method of any of the preceding claims, wherein the
measuring comprises separating phosphorylated and unphosphorylated
iASPP, and measuring iASPP phosphorylation by measuring the amounts
of separated phosphorylated and unphosphorylated iASPP.
25. The method of claim 24, wherein the separating comprises gel
electrophoresis.
26. The method of claim 4, wherein the measuring comprises
isolating iASPP from the cancer cell, and measuring phosphorylated
serine in the isolated iASPP.
27. The method of claim 4, wherein the diagnosing comprises
comparing the amount of phosphorylated iASPP in the cancer cell
with a measurement of phosphorylated iASPP from healthy cells of
the same type as the cancer cell, wherein increased iASPP
phosphorylation in the cancer cell indicates that the cancer is at
increased risk for metastases.
28. The method of claim 4, wherein the diagnosing comprises
comparing the percentage of iASPP that is phosphorylated in the
cancer cell with a calculation of the percentage of iASPP that is
phosphorylated in a healthy cell of the same type as the cancer
cell, wherein increased percentage of phosphorylated iASPP in the
cancer cell indicates that the cancer is at increased risk for
metastases.
29. The method of claim 8, wherein the iASPP polypeptide or the
binding partner is attached to a solid support, and binding is
detected by detecting a complex between the polypeptide and the
binding partner on the solid support.
30. The method of claim 8, wherein the binding partner comprises a
detectable label, and wherein binding is detected by detecting the
label in a complex between the iASPP polypeptide and the binding
partner.
31. The method of claim 1, wherein the measuring comprises
measuring intracellular localization of iASPP in the cancer
cell.
32-34. (canceled)
35. The method of claim 31, wherein the measuring of the
intracellular localization comprises at least one of: an analytical
technique that provides an absolute measure of iASPP in the nucleus
of the cancer cell, an analytical technique that provides a
relative measure of iASPP in the nucleus to total iASPP in the
cell, and an analytical technique that provides a relative measure
of iASPP in the nucleus to iASPP in the cytoplasm of the cell.
36. The method of claim 31, comprising comparing the measure of
iASPP in the nucleus of the cancer cell with a measure of iASPP in
a healthy cell of the same type as the cancer cell, wherein
increased iASPP in the nucleus of the cancer cell compared to the
healthy cells indicates an increased metastatic potential for the
cancer.
37. The method of claim 36, wherein the measuring comprises
immunohistochemical staining of the cancer cell with an antibody
that specifically binds iASPP.
38. The method of claim 37, further comprising contacting the
cancer cell with a nucleus-specific marker.
39. The method of claim 1, comprising repeating the measuring in a
plurality of cancer cells from the subject, wherein an increased
percentage of cancer cells with phosphorylated iASPP or
nuclear-localized iASPP indicates an increased metastatic potential
for the cancer.
40. The method of claim 1, wherein the cancer cell is from a cancer
selected from the group consisting of melanoma, prostate cancer and
head and neck cancer.
41. The method of claim 1, further comprising measuring the mitotic
index of the cancer.
42-43. (canceled)
44. An iASPP binding partner selected from the group consisting of:
(a) antibody that specifically binds iASPP polypeptide and
preferentially binds to iASPP having a phosphorylated serine
compared to binding to iASPP without the phosphorylated serine,
wherein said serine residue corresponds to a serine selected from
the group consisting of Ser84 and Ser113 of the human iASPP amino
acid sequence of SEQ ID NO: 2; (b) a fragment of (a) that
specifically binds iASPP polypeptide and preferentially binds to
iASPP having the phosphorylated serine; and (c) a polypeptide that
comprises (b) and that specifically binds iASPP polypeptide and
preferentially binds to iASPP having the phosphorylated serine.
45-52. (canceled)
53. An iASPP binding partner selected from the group consisting of:
(a) antibody that specifically binds iASPP polypeptide and
preferentially binds to iASPP having an unphosphorylated serine
compared to binding to iASPP with the phosphorylated serine,
wherein said serine residue corresponds to a serine selected from
the group consisting of Ser84 and Ser113 of the human iASPP amino
acid sequence of SEQ ID NO: 2; (b) a fragment of (a) that
specifically binds iASPP polypeptide and preferentially binds to
iASPP having the unphosphorylated serine; and (c) a polypeptide
that comprises (b) and that specifically binds iASPP polypeptide
and preferentially binds to iASPP having the unphosphorylated
serine.
54-66. (canceled)
67. An isolated peptide that comprises an amino acid sequence
selected from the group consisting of: (a) a fragment of the iASPP
amino acid sequence set forth in SEQ ID NO: 2, wherein the fragment
binds to a kinase that phosphorylates iASPP at a serine residue
selected from the group consisting of Ser84 and Ser113 of SEQ ID
NO: 2; (b) an analog of (a) wherein from 1-10 amino acids have been
deleted, inserted, or replaced with different amino acids, wherein
said analog binds to the kinase that phosphorylates iASPP at a
serine residue selected from the group consisting of Ser84 and
Ser113 of SEQ ID NO: 2; (c) a cyclized version of (a) or (b) that
binds to the kinase that phosphorylates iASPP at a serine residue
selected from the group consisting of Ser84 and Ser113 of SEQ ID
NO: 2; wherein the peptide binds to the kinase that phosphorylates
iASPP at a serine residue selected from the group consisting of
Ser84 and Ser113 of SEQ ID NO: 2, and wherein the peptide inhibits
the kinase from phosphorylating iASPP.
68. An isolated peptide that comprises an amino acid sequence that
is at least 90% identical to the amino acid sequence set forth in
SEQ ID NO: 2 or a fragment thereof, wherein the peptide binds to a
kinase that phosphorylates iASPP at a serine residues selected from
the group consisting of Ser84 and Ser113 of SEQ ID NO: 2 and
wherein the peptide inhibits the kinase from phosphorylation of the
serine residue.
69-73. (canceled)
74. An isolated peptide that comprises an amino acid sequence at
least 95% or more identical to amino acids 1-240 of SEQ ID NO: 2,
or a fragment thereof that inhibits translocation of iASPP into the
nucleus of a cancer cell.
75. (canceled)
76. A therapeutic regimen for treating a cancer in a mammalian
subject, the method comprising: (a) measuring phosphorylation state
and/or intracellular localization of inhibitory
Apoptosis-Stimulating Protein of p53 (iASPP) in a cancer cell from
a mammalian subject, (b) screening the cancer cell for at least one
parameter selected from the group consisting of: a p53 mutation
that reduces a tumor suppressive function of p53, an elevated
measurement of mdm2 protein or mRNA, and a B-RafV600E mutation; and
(c) for a subject with a cancer characterized by elevated iASPP
phosphorylation and/or iASPP nuclear localization, prescribing
and/or administering a therapeutic regimen that includes an
inhibitor of iASPP phosphorylation.
77-85. (canceled)
86. A system or identifying metastatic potential of a cancer in a
human subject, the system comprising: (a) at least one processor;
(b) at least one computer-readable medium; (c) a database
operatively coupled to a computer-readable medium of the system and
containing population information correlating the phosphorylation
state or intracellular localization of iASPP in cancer and
metastasis data in a population of humans; (d) a measurement tool
that receives an input about the human subject and generates
information from the input about the iASPP phosphorylation or iASPP
intracellular localization in a cancer cell from the human subject;
and (e) an analysis tool or routine that: (i) is operatively
coupled to the database and the measurement tool, (ii) is stored on
a computer-readable medium of the system, (iii) is adapted to be
executed on a processor of the system, to compare the information
about the human subject with the population information in the
susceptibility database and generate a conclusion with respect to
susceptibility to metastatic potential of the cancer in the human
subject.
87. (canceled)
88. A composition comprising an inhibitor of cyclin B1/cdk1-induced
phosphorylation of iASPP and a pharmaceutically acceptable
carrier.
89-90. (canceled)
91. A unit dose comprising an inhibitor of cyclin B1/cdk1-induced
phosphorylation of iASPP; an inhibitor of mdm2-induced degradation
of p53; and an inhibitor B-RafV600E; wherein the inhibitors are
packaged together, and at least two of the inhibitors are not in
admixture.
92. (canceled)
93. A method of treating a mammalian subject with cancer, the
method comprising: administering to the subject an inhibitor of
cyclin B1/cdk1-induced phosphorylation of iASPP; an inhibitor of
mdm2-induced degradation of p53; and an inhibitor B-RafV600E;
wherein the inhibitors are administered in amounts effective to
inhibit proliferation of the cancer.
94-98. (canceled)
Description
BACKGROUND OF THE INVENTION
[0001] Melanoma is the most lethal skin tumour, and its incidence
is rising. Metastatic melanoma is refractory to treatment and is
associated with a very poor prognosis. Advanced/metastatic melanoma
frequently harbours mutations in the RAS-BRAF-MAPK oncogene and in
the p16ink4A-Rb and p14Arf-p53 tumour suppressor pathways. Around
20% and 44% of melanomas harbor active mutations of N-RAS and
B-RAF, respectively, and these tumours have elevated RAS/RAF kinase
activities (Forbes et al., 2006). Similarly, deletion in the
p16ink4A locus, which will inactivate both p16ink4a and p14Arf,
also occurs in around 50% of melanomas (Curtin et al., 2005).
P16ink4a is an inhibitor of cyclin dependent kinases, cyclinD1/cdk4
in particular. Melanomas containing p16ink4a mutations will have
elevated cyclinD1/cdk4 kinase activity that will in turn
phosphorylate the tumour suppressor Rb and inactivate its ability
to arrest the cell at G1 phase of the cell cycle. Mutation of
p14Arf will remove its ability to block the action of mdm2, an
inhibitor and E3 ubiquitin ligase of p53. As a result, p14Arf
mutant melanoma cells may have wild type p53, but its tumour
suppressive functions are lost. The importance of RAS-RAF oncogene
and p16ink4a/Rb and p14Arf/p53 tumour suppressor pathways in the
development and maintenance of melanomas is underscored by a
plethora of experimental evidence. This knowledge has led to the
recent success of B-RAF inhibitors, such as PLX4032, as new
melanoma therapeutic agents. PLX4032 selectively inhibits
proliferation of tumour cells that harbour the B-RafV600E mutation
(Joseph et al., 2010). Despite promising efficacy in inhibiting
melanoma growth in the initial stages of treatment, the majority of
PLX4032 treated patients relapsed within a few months, largely due
to acquired drug resistance. As expected, the majority of the drug
resistance was caused by mutations affecting the RAS/MAPK pathways
(Chapman et al., 2011; Cox and Der, 2012; Johannessen et al., 2010;
Nazarian et al., 2010; Poulikakos and Rosen, 2011; Poulikakos and
Solit, 2011). (Chapman et al., 2011; Johannessen et al., 2010;
Nazarian et al., 2010). To overcome acquired drug resistance,
co-targeting B-RAFV600E and MEK has been suggested (Poulikakos
2011). As both B-RAF and MEK are in the same pathway, it is
difficult to predict whether targeting these two components of the
same pathway will be sufficient to eliminate all melanoma cells. An
alternative strategy would be to induce synthetic lethality of
melanoma cells by co-targeting two independent pathways that are
both critical in melanoma development and maintenance.
[0002] The tumor suppressor p53 pathway is of particular interest.
B-RAFV600E is present in around 50% of human melanomas; however,
80% of human melanomas express wild type p53 implying that a large
number of B-RAFV600E melanomas also contain wild type p53. It has
been established that growth suppression induced by B-RAFV600E
inhibitors is independent of p53. It is also known that the tumor
suppressive function of p53 is largely lost in melanoma cells
esspressing wild type p53 expressing tumors.
[0003] Overexpression of mdm2 or and mdmX are two inhibitors of
p53, has been observed in melanomas, and this was shown to
contribute to the inactivation of wild type p53 function (Muthusamy
et al., 2006; Polsky et al., 2001). To restore the tumor
suppressive function of p53 in human tumors, including melanomas
that express wild type p53, agonists of p53 such as Nutlin3 have
been developed and tested in Phase II clinical trials. Nutlin3
suppresses tumor growth by inducing cell cycle arrest through it
ability to prevent mdm2 from targeting p53 for degradation.
Unfortunately, nutlin3 failed to reactivate p53 in all wild type
p53-expressing melanoma cells (de Lange et al., 2011; Ji et al.,
2011). It is therefore important to identify an alternative
strategy for reactivating the tumor suppressing function of p53 in
melanoma cells.
[0004] A need exists for modulators of apoptosis. For example,
pro-apoptotic modulators may be useful for treating or palliating
cancers or other conditions characterized by undesirable or
uncontrolled cell growth. Anti-apoptotic factors may be useful for
treating or palliating degenerative conditions.
SUMMARY OF THE INVENTION
[0005] The present invention has numerous aspects and is based in
part on discoveries described herein involving iASPP molecular and
cell biology, including discoveries including iASPP
phosphorylation, iASPP intracellular localization, the effect of
iASPP phosphorylation and location of metastasis of a cancer cell
and other insights.
[0006] In one aspect of the invention, a method of evaluating
metastatic potential of a cancer is provided. In some variations,
the method comprises (a) measuring phosphorylation state and/or
intracellular localization of inhibitory Apoptosis-Stimulating
Protein of p53 (iASPP) in a cancer cell from a mammalian subject,
and (b) diagnosing (or evaluating or scoring) metastatic potential
of the cancer based on the phosphorylation state and/or
intracellular localization of iASPP in the cancer cell measured
according to step (a). In some embodiments, the methods comprises
measuring phosphorylation state and/or intracellular localization
of iASPP in a plurality of cancer cells from the mammalian subject,
and diagnosing metastatic potential of the cancer based on an
average measurement from the plurality of the cells, or from a
percentage of the cells that exceed a cutoff or reference
measurement. The diagnosing of metastatic potential is optionally
made from the percentage of total iASPP that is phosphorylated or
the percentage of total iASPP that is localized in the nucleus of
the cell.
[0007] Without intending to limit the invention to any particular
theory, it appears that iASPP phosphorylation and iASPP
localization to the nucleus are biologically related, and both
phenomena are useful--if not interchangeable--indicators of the
metastatic potential of a cancer. Therefore, if an aspect of the
invention is described in the context of iASPP phosphorylation, the
invention should be understood to include a related embodiment
involving iASPP intracellular localization, and vice versa. For
brevity, not all aspects have been described in duplicate with
respect to these phenomena.
[0008] In any of the methods described herein, the mammalian
subject is preferably human. Coding and deduced amino acid
sequences for a wild type human iASPP are set forth in SEQ ID NO: 1
and 2, respectively.
[0009] In some embodiments, the cancer cell is from a primary tumor
from the subject. For example, the cell(s) may be obtained from a
biopsy, of following tumor resection from a patient.
[0010] The diagnostic methods described herein optionally comprise
screening the cancer cell for at least one parameter selected from
the group consisting of: a p53 mutation that reduces a tumor
suppressive function of p53, an elevated measurement of mdm2
protein or mRNA, and a B-RafV600E mutation; and diagnosing
metastatic potential of the cancer based on the phosphorylation
state and/or intracellular localization of iASPP in the cancer cell
and from the presence or absence of the at least one parameter. In
some embodiments, the at least one parameter comprises the p53
mutation that reduces a tumor suppressive function of p53.
[0011] The data collected relating to these parameters has
relevance to the selection of a course of therapy for the subject
with the cancer, as described in greater detail below. Methods that
include prescribing or administering a therapy based on the data
obtained with respect to these parameters also is an aspect of the
invention.
[0012] The measuring of phosphorylated iASPP can occur after a
cancer diagnosis has been made and prior to in initiation of a
standard of care cancer therapy (e.g., chemotherapy). In some
embodiments, the measuring of phosphorylated iASPP occurs after a
cancer has become resistant to a standard of care therapy (e.g.,
chemotherapy). These embodiments are not mutually exclusive. A
subject undergoing cancer therapy can be monitored for
phosphorylated iASPP expression to identify a time point at which
phosphorylated iASPP becomes elevated and therapy with a cdk1
inhibitor is indicated according to the invention.
[0013] In some embodiments, measuring of the phosphorylation state
comprises at least one of: an analytical technique that provides an
absolute measure of phosphorylated iASPP; an analytical technique
that provides a relative measure of phophorylated iASPP to total
iASPP in the cell; and an analytical technique that provides a
relative measure of phosphorylated iASPP to unphosphorylated iASPP
in the cell.
[0014] To assess the relative level of phosphorylated iASPP
expression, the level of phosphorylated iASPP expression in a
cancer tissue sample can be subjected to one or more of various
comparisons. In general, it can be compared to: (a) phosphorylated
iASPP expression level(s) in normal tissue from the organ in which
the cancer originated; (b) phosphorylated iASPP expression level(s)
in a collection of comparable cancer tissue samples; (c)
phosphorylated iASPP expression level(s) in a collection of normal
tissue samples; or (d) phosphorylated iASPP expression level(s) in
an arbitrary standard. In some embodiments, the screening methods
described herein comprises comparing the expression of
phosphorylated iASPP in the tumor (or vessel tissue or tumor fluid)
to the level of phosphorylated iASPP expression in healthy tissue
of the same type as the tumor, wherein elevated phosphorylated
iASPP expression in the tumor (or vessel tissue or tumor fluid) to
compared to the healthy tissue identifies the metastatic potential
of the cancer.
[0015] In some embodiments, the measuring comprises measuring the
phosphorylation state of iASPP in the cancer cell, wherein the
phosphorylation is serine phosphorylation at an iASPP serine
residue corresponding to serine 84 (Ser84) and/or serine 113
(Ser113) in the human iASPP amino acid sequence of SEQ ID NO:
2.
[0016] The measuring step of the methods described herein
optionally comprises comparing the measurement of phosphorylated
iASPP to a reference measurement of phosphorylated iASPP, and
scoring the phosphorylated iASPP measurement from the sample as
elevated based on statistical analysis or a ratio relative to the
reference measurement. In some embodiments, the reference
measurement comprises at least one of the following (a) a
measurement of phosphorylated iASPP from healthy tissue of the
subject of the same tissue type as the sample; (b) a database
containing multiple phosphorylated iASPP measurements from healthy
or cancerous tissues from other subjects; or (c) a reference value
calculated from multiple phosphorylated iASPP measurements from
healthy or cancerous tissues from other subjects, optionally
further including statistical distribution information for the
multiple measurements, such as standard deviation.
[0017] In some embodiments, a phosphorylated iASPP measurement of
at least 1.0 standard deviation greater than a median
phosphorylated iASPP measurement in corresponding healthy tissue is
scored as elevated phosphorylated expression. In other embodiments,
a phosphorylated measurement that is statistically significantly
greater than phosphorylated iASPP measurements in corresponding
healthy tissue, with a p-value less than 0.1 is scored as elevated
phosphorylated iASPP expression.
[0018] In some variations, the level of phosphorylated iASPP in a
subject is compared to a predetermined "cut-off" concentration of
phosphorylated iASPP that has been determined from observations to
represent an elevated measure of phosphorylated iASPP that (when
equaled or exceeded) is predictive of metastatic potential in the
cancer. Determination of a suitable cut-off is made using, e.g.,
statistical analysis of phosphorylated iASPP concentration data
collected from multiple healthy and/or cancer patients. If a
"cut-off" value is employed, the cut-off concentration preferably
is statistically determined to have optimal discriminating value to
identify cancer with metastatic potential. It will be appreciated
that statistical analysis of a dataset will permit clinicians to
make informed decisions based on concentrations other than the
optimal discriminating concentration (e.g., above or below the
optimal discriminating concentration). For example, using
receiver-operating-characteristic curves, or using other
statistical summaries of phosphorylated iASPP concentration and
metastases outcome data collected according to the invention, the
practitioner is capable of selecting a cut-off phosphorylated iASPP
concentration having a desired level of sensitivity or specificity
for predicting the metastatic potential of the cancer.
[0019] The output or conclusion from methods as described here are
not limited to binary classification of tumors having (or not
having) metastatic potential. Cancers can be stratified into two,
three, four, five, or more categories of metastatic risk potential,
based on the measurement of iASPP phosphorylation (or iASPP
intracellular localization). For example, the percent of total
iASPP that is phosphorylated, or localized to the nucleus, can be
used to stratify the cancers.
[0020] A variety of techniques can be used to measure iASPP, and to
measure iASPP phosphorylation or intracellular localization, and
all such techniques are contemplated for practicing the
invention.
[0021] The diagnostic methods described herein optionally comprises
contacting the cancer cell, or protein isolated from the cancer
cell, with a binding partner that specifically binds phosphorylated
iASPP, and measuring the phosphorylation state of iASPP by
measuring the amount of the binding partner-iASPP complex formed.
In some embodiments, the binding partner comprises a detectable
label. In some embodiments, the diagnostic methods comprise
contacting the cancer cell, or protein isolated from the cancer
cell, with a first binding partner and a second binding partner,
wherein the first binding partner binds iASPP and the second
binding partner specifically binds the phosphorylated iASPP,
wherein the second binding partner comprises a detectable label,
and measuring the phosphorylation state of iASPP by measuring the
amount of second binding partner-iASPP complex formed. The
phosphorylated iASPP recognized by the second binding partner
optionally comprises one or more phosphorylated serine residues. In
some embodiments, the serine residues are selected from the group
consisting of Ser84 and Ser113. In some embodiments, the first
binding partner is an antibody that binds to an epitope comprising
all or part of, or contained within, amino acids 501-510 of SEQ ID
NO: 2 and the second binding partner is an antibody that binds to
an epitope comprising all or part of, or contained within, amino
acids 107-118 of SEQ ID NO: 2, wherein the amino acid at position
113 of SEQ ID NO: 2 in the epitope is phosphorylated.
[0022] In some embodiments, the diagnostic methods comprise
contacting the cancer cell, or protein isolated from the cancer
cell, with a first binding partner and a second binding partner
that binds iASPP, wherein the first binding partner binds
phosphorylated and unphosphorylated iASPP, and wherein the second
binding partner binds an epitope of iASPP that includes Ser84 or
Ser113 of iASPP (SEQ ID NO: 2) and selectively binds iASPP when
said serine in said epitope is unphosphorylated, and wherein the
second binding partner comprises a detectable label, and measuring
the phosphorylation state of iASPP by measuring the amount of
second binding partner-iASPP complex formed. In some embodiments,
the first binding partner is an antibody that binds to an epitope
comprising all or part of, or contained within, amino acids 501-510
of SEQ ID NO: 2 and the second binding partner is an antibody that
binds to an epitope comprising all or part of, or contained within,
amino acids 111-122 of SEQ ID NO: 2.
[0023] In some embodiments, the diagnostic methods comprise
measuring total iASPP in a cell. For example, in some embodiments,
the method comprises measuring total iASPP in the cell, and
measuring unphosphorylated iASPP in the cell, wherein a difference
between total iASPP and unphosphorylated iASP provides an
indication of the amount of phosphorylated iASPP in the cell. The
method optionally comprises measuring unphosphorylated iASPP in the
cell comprises contacting the cancer cell, or protein isolated from
the cancer cell, with a binding partner that preferentially binds
iASPP in which one or more serine residues selected from serine 84
(Ser84, SEQ ID NO: 2) and serine 113 (Ser113, SEQ ID NO: 2) is not
phosphorylated. An exemplary antibody that recognizes an
unphosphorylated epitope is antibody LX128.5.
[0024] Measuring iASPP phosphorylation in a cell comprises, in some
embodiments, contacting the cancer cell, or protein isolated from
the cancer cell, with a binding partner that preferentially binds
iASPP in which one or more serine residues selected from serine 84
(Ser84, SEQ ID NO: 2) and serine 113 (Ser113, SEQ ID NO: 2) is
phosphorylated, and measuring the phosphorylation state of iASPP by
measuring the amount of polypeptide-binding partner complex formed,
to provide a measure of iASPP that is phosphorylated at one or more
of the serine residues. In such embodiments, the binding partner is
an antibody that preferentially recognizes an iASPP epitope in
which one or more serine residues selected from serine 84 (Ser84,
SEQ ID NO: 2) and serine 113 (Ser113, SEQ ID NO: 2) is
phosphorylated, compared to epitopes of iASPP in which Ser84 and
Ser113 are unphosphorylated. In some embodiments, the binding is
measured in situ in a tumor biopsy.
[0025] In another aspect, described herein is a method of measuring
iASPP phosphorylation state in a cancer cell from a mammalian
subject, wherein the method comprises contacting the cancer cell,
or a sample derived from the cancer cell (such as a cell lysate or
cellular fraction, or protein isolated from the cancer cell, with a
binding partner that specifically binds phosphorylated iASPP, and
measuring the phosphorylation state of iASPP by measuring the
amount of the binding partner-iASPP complex formed. In some
embodiments, the binding partner comprises a detectable label. In
some embodiments, the assaying method comprises contacting the
cancer cell, or the sample derived from the cell, or the protein
isolated from the cancer cell, with a first binding partner and a
second binding partner, wherein the first binding partner binds
iASPP and the second binding partner specifically binds the
phosphorylated iASPP, wherein the second binding partner comprises
a detectable label, and measuring the phosphorylation state of
iASPP by measuring the amount of second binding partner-iASPP
complex formed (e.g., using a procedure that includes measuring the
detectable label). The phosphorylated iASPP recognized by the
second binding partner optionally comprises one or more
phosphorylated serine residues. In some embodiments, the serine
residues are selected from the group consisting of Ser84 and
Ser113. In some embodiments, the first binding partner is an
antibody that binds to an epitope comprising all or part of, or
contained within, amino acids 501-510 of SEQ ID NO: 2 and the
second binding partner is an antibody that binds to an epitope
comprising all or part of, or contained within, amino acids 107-118
of SEQ ID NO: 2, wherein the amino acid at position 113 of SEQ ID
NO: 2 is phosphorylated in the epitope.
[0026] In some embodiments, the assaying methods comprise
contacting the cancer cell, or a biological sample derived from the
cancer cell (such as a cell lysate or subcellular fraction, or
protein isolated from the cancer cell, with a first binding partner
and a second binding partner that binds iASPP, wherein the first
binding partner binds phosphorylated and unphosphorylated iASPP,
and wherein the second binding partner binds an epitope of iASPP
that includes Ser84 or Ser113 of iASPP (SEQ ID NO: 2) and
selectively binds iASPP when said serine in said epitope is
unphosphorylated, and wherein the second binding partner comprises
a detectable label, and measuring the phosphorylation state of
iASPP by measuring the amount of second binding partner-iASPP
complex formed. In some embodiments, the first binding partner is
an antibody that binds to an epitope comprising all or part of, or
is contained within, amino acids 501-510 of SEQ ID NO: 2 and the
second binding partner is an antibody that binds to an epitope
comprising all or part of, or is contained within, amino acids
111-122 of SEQ ID NO: 2.
[0027] The diagnostic or assaying methods described herein
optionally include a separation step to determine a measurement of
phosphorylated iASPP in a sample. In such embodiments, the
measuring comprises separating phosphorylated and unphosphorylated
iASPP, and measuring iASPP phosphorylation by measuring the amounts
of separated phosphorylated and unphosphorylated iASPP. The
separating can be performed by any means known in the art, such as
gel electrophoresis. In some embodiments, the measuring comprises
isolating iASPP from the cancer cell, and measuring phosphorylated
serine in the isolated iASPP.
[0028] The diagnostic or assaying methods described herein
optionally comprise the step of making a communication that
includes metastatic potential of the cancer based on the
phosphorylation state and/or intracellular localization of iASPP in
the cancer cell available to the individual or to a third party. In
some embodiments, the communication is made available to the
individual or third party by a secured internet interface.
Exemplary third party include, but are not limited to a physician
or another health care worker, or an insurer or health care payer
government or private entity.
[0029] As described herein and depicted in figures, phosphorylated
iASPP appears to migrate at a different speed in gel
electrophoresis. Thus, in some variations, the measurement of
amounts of fast- and slow-migrating forms of iASPP is used as a
tool for measuring the amount of iASPP phosphorylation from a cell
or cell lysate. This can be accomplished, for example, by gel
electrophoresis and western blotting.
[0030] In various embodiments, the diagnosing comprises comparing
the amount of phosphorylated iASPP in the cancer cell with a
measurement of phosphorylated iASPP from healthy cells of the same
type as the cancer cell, wherein increased iASPP phosphorylation in
the cancer cell indicates that the cancer is at increased risk for
metastases. In some embodiments, the diagnosing comprises comparing
the percentage of iASPP that is phosphorylated in the cancer cell
with a calculation of the percentage of iASPP that is
phosphorylated in a healthy cell of the same type as the cancer
cell, wherein increased percentage of phosphorylated iASPP in the
cancer cell indicates that the cancer is at increased risk for
metastases.
[0031] In various embodiments, the iASPP polypeptide or the binding
partner is attached to a solid support, and binding is detected by
detecting a complex between the polypeptide and the binding partner
on the solid support. The binding partner optionally comprises a
detectable label, and wherein binding is detected by detecting the
label in a complex between the iASPP polypeptide and the binding
partner.
[0032] The diagnostic methods, in various embodiments, comprise
measuring intracellular localization of iASPP in the cancer cell,
wherein the measuring comprises at least one of an analytical
technique that provides an absolute measure of iASPP in the nucleus
of the cancer cell, an analytical technique that provides a
relative measure of iASPP in the nucleus to total iASPP in the
cell, and an analytical technique that provides a relative measure
of iASPP in the nucleus to iASPP in the cytoplasm of the cell. In
some embodiments, the method comprises comparing the measure of
iASPP in the nucleus of the cancer cell with a measure of iASPP in
a healthy cell of the same type as the cancer cell, wherein
increased iASPP in the nucleus of the cancer cell compared to the
healthy cells indicates an increased metastatic potential for the
cancer.
[0033] Measuring intracellular localization of iASPP can be
performed by methods known in the art, such as
immunohistochemistry. For example, the intracellular localization
of iASPP can be measured by immunohistochemical staining of the
cancer cell with an antibody that specifically binds iASPP. In some
embodiments, the measuring further comprises contacting the cancer
cell with a nucleus-specific marker.
[0034] For any of the diagnostic methods described herein,
repeating the measuring steps is specifically contemplated. For
example, in some embodiments, the method comprises repeating the
measuring in a plurality of cancer cells from the subject, wherein
an increased percentage of cancer cells with phosphorylated iASPP
or nuclear-localized iASPP indicates an increased metastatic
potential for the cancer.
[0035] In various embodiments, the diagnostic methods further
comprise measuring the mitotic index of the cancer.
[0036] Any available technique can be used for measuring
phosphorylated iASPP expression, including direct and indirect
techniques. Exemplary techniques for measuring amounts or
concentrations of phosphorylated iASPP in a sample are
immunological techniques that involve use of a polyclonal or
monoclonal antibody that specifically binds phosphorylated iASPP,
or use of a phosphorylated iASPP-binding fragment of such an
antibody. For example, the measuring comprises contacting the
biological sample with a iASPP antibody that preferentially binds a
phosphorylated form of iASPP or antigen-binding fragment thereof.
Quantification of the amount of bound antibody (e.g., using a label
or second, labeled antibody) provides a measurement of
phosphorylated iASPP expressed in the sample. The use of
immunoassays such as radioimmunoassay, immunoradiometric assay
(labeled antibody), or an enzyme-linked immunosorbent assay (ELISA)
are contemplated.
[0037] The screening methods described herein may optionally
comprise the step of prescribing for or administering to the
subject identified as having elevated phosphorylated iASPP
expression in the biological sample a composition comprising a
molecule that suppresses expression or phosphorylation activity of
cyclinB1/cdk1. Data presented herein implicate cyclinB1/cdk1 as a
kinase responsible for phosphorylation of iASPP. The human cDNA and
deduced amino acid sequences for this enzyme are set forth in
Genbank Accession No. NM.sub.--001786 and Swiss/Prot Accession Nos.
P06943, respectively.
[0038] Another aspect of the invention is an iASPP binding partner
selected from the group consisting of (a) antibody that
specifically binds iASPP polypeptide and preferentially binds to
iASPP having a phosphorylated serine compared to binding to iASPP
without the phosphorylated serine, wherein said serine residue
corresponds to a serine selected from the group consisting of Ser84
and Ser113 of the human iASPP amino acid sequence of SEQ ID NO: 2;
(b) a fragment of (a) that specifically binds iASPP polypeptide and
preferentially binds to iASPP having the phosphorylated serine; and
(c) a polypeptide that comprises (b) and that specifically binds
iASPP polypeptide and preferentially binds to iASPP having the
phosphorylated serine. The binding partner optionally binds to an
epitope of the phosphorylated peptide comprising a portion of amino
acids 81-130 of SEQ ID NO: 2, wherein one or both of the serine
residues as positions 84 and 113 of SEQ ID NO: 2 are
phosphorylated. In some embodiments, the binding partner binds to
an epitope of the phosphorylated peptide comprising all or part
(some) of amino acids 107-118 of SEQ ID NO: 2, wherein the serine
at position 113 of SEQ ID NO: 2 is phosphorylated.
[0039] In some embodiments, the binding partner is an antibody or
antibody fragment. In some embodiments, the antibody or antibody
fragment (including, but not limited to, Fab, Fab', F(ab')2, ScFv
and Fv fragments) does not bind to unphosphorylated iASPP.
[0040] Also provided is an iASPP binding partner selected from the
group consisting of (a) antibody that specifically binds iASPP
polypeptide and preferentially binds to iASPP having an
unphosphorylated serine compared to binding to iASPP with the
phosphorylated serine, wherein said serine residue corresponds to a
serine selected from the group consisting of Ser84 and Ser113 of
the human iASPP amino acid sequence of SEQ ID NO: 2; (b) a fragment
of (a) that specifically binds iASPP polypeptide and preferentially
binds to iASPP having the unphosphorylated serine; and (c) a
polypeptide that comprises (b) and that specifically binds iASPP
polypeptide and preferentially binds to iASPP having the
unphosphorylated serine. In some embodiments, the iASPP binding
partner binds to an epitope of the unphosphorylated iASPP that
comprises all or some of, or is contained within amino acids
111-122 of SEQ ID NO: 2. Antibody LX128.5 is an exemplary antibody
that preferentially recognizes an epitope of the N-terminus of
unphosphorylated iASPP.
[0041] Another aspect is a kit comprising a first antibody that
recognizes an iASPP polypeptide having at least one phosphorylated
serine residue selected from the group consisting of Ser84 and
Ser113 and optionally a second antibody that binds to iASPP at an
epitope distinct from that recognized by the first antibody. The
first antibody, in some embodiments, binds to an epitope comprising
all or some of amino acids 107-118 of SEQ ID NO: 2, wherein the
serine at position 113 of SEQ ID NO: 2 is phosphorylated in the
epitope. In some embodiments, the second antibody binds
phosphorylated and unphosphorylated iASPP. The second antibody, in
some embodiments, binds to an epitope comprising all or some of
amino acids 501-510 of SEQ ID NO: 2. In some embodiments, the kit
further comprises a nucleus specific marker.
[0042] Another aspect is a kit comprising a first antibody and a
second antibody, wherein the first antibody recognizes an iASPP
polypeptide and the second antibody binds an epitope of iASPP that
includes Ser84 or Ser113 of iASPP (SEQ ID NO: 2) and selectively
binds iASPP when said serine in said epitope is unphosphorylated,
and wherein the second antibody optionally comprises a detectable
label, such as a radioisotope or a fluorophor or an enzyme or
enzyme substrate. In some embodiments, the second antibody binds to
iASPP at an epitope distinct from that recognized by the first
antibody. In some embodiments, the first antibody binds to an
epitope comprising all or some of amino acids 501-510 of SEQ ID NO:
2 and the second antibody binds to an epitope comprising all or
some of amino acids 111-122 of SEQ ID NO: 2. The kit optionally
comprises a nucleus-specific marker.
[0043] In some variations, iASPP binding partners described herein
are antibodies (including antibody fragments and engineered
antibody molecules). For example, the invention includes:
[0044] A. An antibody that specifically binds to an epitope of
human iASPP (SEQ ID NO: 2), wherein the epitope includes a serine
of the iASPP at position 84 or 113 of SEQ ID NO: 2, and wherein the
antibody preferentially or selectively or specifically binds the
iASPP when the serine in the epitope is unphosphorylated.
[0045] A1. The antibody according to paragraph A that comprises a
monoclonal antibody or comprises an antigen-binding fragment
thereof.
[0046] A2. The antibody according to paragraph A or A1 that further
comprises a detectable label.
[0047] A3. The antibody according to any one of paragraphs A-A2,
wherein the epitope includes the serine at position 84 of SEQ ID
NO: 2 (Ser84).
[0048] A4. The antibody according to paragraph A or A1, wherein the
epitope includes the serine at position 113 of SEQ ID NO: 2
(Ser113).
[0049] A5. The antibody according to paragraph A4, wherein the
epitope comprises or is contained within amino acids 107-118 of SEQ
ID NO: 2.
[0050] A6. A composition comprising the antibody according to any
one of paragraphs A-A4 that is substantially free of antibody that
binds the iASPP when the serine is phosphorylated.
[0051] A7. The composition according to paragraph A6 that includes
the antibody that binds to the epitope that includes Ser84 and the
antibody that binds to the epitope that includes Ser113.
[0052] In related variations, the invention includes:
[0053] B. An antibody that specifically binds to an epitope of
human iASPP (SEQ ID NO: 2), wherein the epitope includes a serine
of the iASPP at position 84 or 113 of SEQ ID NO: 2, and wherein the
antibody preferentially or selectively or specifically binds the
iASPP when the serine in the epitope is phosphorylated.
[0054] B1. The antibody according to paragraph B that comprises a
monoclonal antibody or comprises an antigen-binding fragment
thereof.
[0055] B2. The antibody according to paragraph B or B1 that further
comprises a detectable label.
[0056] B3. The antibody according to any one of paragraphs B-B2,
wherein the epitope includes the serine at position 84 of SEQ ID
NO: 2 (Ser84), wherein the serine is phosphorylated.
[0057] B4. The antibody according to paragraph B or B1, wherein the
epitope includes the serine at position 113 of SEQ ID NO: 2
(Ser113), wherein the serine is phosphorylated.
[0058] B5. The antibody according to paragraph B4, wherein the
epitope comprises or is contained within amino acids 107-118 of SEQ
ID NO: 2, wherein the serine is phosphorylated.
[0059] B6. A composition comprising the antibody according to any
one of paragraphs B-B4 that is substantially free of antibody that
binds the iASPP when the serine is unphosphorylated.
[0060] B7. The composition according to paragraph B6 that includes
the antibody that binds to the epitope that includes phosphorylated
Ser84 and the antibody that binds to the epitope that includes
phosphyrlated Ser113.
[0061] As described elsewhere herein, the invention includes kits
in which iASPP binding partners are packaged with each other and/or
with other reagents that are useful together for evaluating iASPP
phosphorylation status. For example the invention includes:
[0062] C. A kit containing the antibody or composition according to
any one of paragraphs A-A7 and B-B7.
[0063] C1. The kit according to paragraph C that further includes a
capture antibody that binds an epitope of human iASPP that is
present in the iASPP irrespective of whether the serines at
positions 84 and 113 are phosphorylated.
[0064] C2. The kit according to paragraph C1, wherein the capture
antibody binds an epitope of iASPP that comprises or is contained
within amino acids 501-510 of SEQ ID NO: 2.
[0065] C3. The kit according to paragraph C1 or C2, wherein the
capture antibody is bound to a solid support.
[0066] C4. The kit according to any one of paragraphs C to C3 that
includes an antibody according to any one of paragraphs A-A7 and an
antibody according to any one of paragraphs B-B7.
[0067] C5. The kit according to any one of paragraphs C to C3 that
includes the antibody according to paragraph A3 and the antibody
according to paragraph A4 or A5.
[0068] C6. The kit according to any one of paragraphs C to C3 and
C5 that includes the antibody according to paragraph B3 and the
antibody according to paragraph B4 or B5.
[0069] C7. The kit according to any one of paragraphs C to C6
further comprising one or more compositions selected from wash
reagents, detecting reagents useful for detecting and/or measuring
a bound antibody (especially measuring a labeled antibody bound to
its target); and a cell nucleus-specific marker.
[0070] C8. The kit according to any one of paragraphs C to C7
wherein the elements are packaged together but not in
admixture.
[0071] The iASPP phosphorylation assays described herein are
themselves an aspect of the invention. As taught in detail herein,
the results of such assays can be used by the party performing the
assays, or a third party, to evaluate cancer status of a subject
and for selection, monitoring, and changing treatment approaches.
Likewise, the results of such assays are useful in a laboratory and
research context for characterizing cancer cell lines, monitoring
changes in cancer cell lines, and evaluating therapeutic compound
candidates against cancer cell lines having different iASPP
phosphorylation characteristics. Thus, aspects of the invention
include iASPP phosphorylation assays such as the following:
[0072] D. A method of measuring a phosphorylation state of iASPP
comprising: contacting iASPP protein with the antibody or
composition according to any one of paragraphs A-A6 and B-B6, and
measuring the amount of antibody that is bound to the iASPP or
measuring the amount of antibody-iASPP complex.
[0073] D1. The method according to paragraph D, wherein the iASPP
is in a biological sample, and the method comprises contacting the
biological sample with the antibody or composition.
[0074] D2. The method according to paragraph D1, wherein the
biological sample is from a cancer cell or a tumor from a mammalian
subject.
[0075] D3. The method according to any one of paragraphs D1-D2,
further comprising measuring the total iASPP in the sample.
[0076] D4. The method according to paragraph D3, further comprising
comparing the measurement of total iASPP with the measurement of
phosphorylated iASPP and/or the measurement of unphosphorylated
iASPP to derive a measurement of the percentage of total iASPP that
is phosphorylated or unphosphorylated.
[0077] D5. The method according to any one of paragraphs D2-D4,
comprising repeating the method using a biological sample from a
non-cancerous cell or tissue of the same tissue type as the cancer
cell or the tumor, and comparing the measures of iASPP
phosphorylation in the cancer versus non-cancerous cells.
[0078] D6. The method according to any one of paragraphs D-D5,
comprising performing the measuring repeatedly with a plurality of
cancer cells from the mammalian subject and generating an average
measurement of other statistical analysis from the repeated
measurements.
[0079] D7. The method according to any one of paragraphs D-D5,
comprising performing the measuring with a lysate from a plurality
of cancer cells from the mammalian subject.
[0080] D8. The method according to any one of paragraphs D1 to D7,
further comprising screening the cancer cells for evidence of a p53
mutation.
[0081] Inhibitor peptides are also considered an aspect of the
invention. For example, in some embodiments, the inhibitor peptide
is an isolated peptide that comprises an amino acid sequence
selected from the group consisting of (a) a fragment of the iASPP
amino acid sequence set forth in SEQ ID NO: 2, wherein the fragment
binds to a kinase that phosphorylates iASPP at a serine residue
selected from the group consisting of Ser84 and Ser113 of SEQ ID
NO: 2; (b) an analog of (a) wherein from 1-10 amino acids have been
deleted, inserted, or replaced with different amino acids, wherein
said analog binds to the kinase that phosphorylates iASPP at a
serine residue selected from the group consisting of Ser84 and
Ser113 of SEQ ID NO: 2; (c) a cyclized version of (a) or (b) that
binds to the kinase that phosphorylates iASPP at a serine residue
selected from the group consisting of Ser84 and Ser113 of SEQ ID
NO: 2; wherein the peptide binds to the kinase that phosphorylates
iASPP at a serine residue selected from the group consisting of
Ser84 and Ser113 of SEQ ID NO: 2, and wherein the peptide inhibits
the kinase from phosphorylating iASPP. In some embodiments, the
kinase is cyclinB1/cdk1.
[0082] In some embodiments, the isolated peptide comprises an amino
acid sequence that is at least 90% identical to the amino acid
sequence set forth in SEQ ID NO: 2 or a fragment thereof, wherein
the peptide binds to a kinase that phosphorylates iASPP at a serine
residues selected from the group consisting of Ser84 and Ser113 of
SEQ ID NO: 2 and wherein the peptide inhibits the kinase from
phosphorylation of the serine residue. In some embodiments, the
peptide variants of the invention do not include S84D or S113D
mutations. Data presented herein shows that iASPP with these
mutations mimics phosphorylated wildtype iASPP.
[0083] In some embodiments, the isolated peptide comprises a
fragment of SEQ ID NO: 2 that optionally includes or consists of
amino acids 1-240 of SEQ ID NO: 2 or amino acids 81-130 of SEQ ID
NO: 2 or amino acids 107-118 of SEQ ID NO: 2.
[0084] Also provided is an isolated peptide that comprises an amino
acid sequence at least 95% or more identical to amino acids 1-240
of SEQ ID NO: 2, or a fragment thereof that inhibits translocation
of iASPP into the nucleus of a cancer cell. In some embodiments,
the peptide variants of the invention do not include S84D or S113D
mutations. Data presented herein shows that iASPP with these
mutations mimics phosphorylated wildtype iASPP.
[0085] A composition comprising any of the isolated peptides
described herein and a pharmaceutically acceptable carrier are also
contemplated.
[0086] Another aspect of the invention is a method of treating a
subject with cancer. For example, a method of the invention
comprises administering to a subject with cancer an agent that
inhibits cdk1 phosphorylation of iASPP. In some variations, the
administering step is preceded by a diagnostic step or method as
described herein, that identifies the subject as having a cancer
with elevated metastatic potential due to iASPP phosphorylation or
iASPP nuclear localization.
[0087] Aspects of the invention that are described herein as
methods (especially methods that involve treatment) can
alternatively be described as (medical) uses of reagents or
therapeutics. For example, in one variation, the invention is a use
of a composition that comprises a molecule that suppresses
expression or activity of cdk1 for the treatment of cancer in a
subject identified with cancer and identified with elevated
phosphorylated iASPP expression in the cancer (wherein the subject
is identified as having elevated phosphorylated iASPP expression in
the cancer by a method described herein).
[0088] In the treatment methods (or uses) described herein, the
methods optionally comprises administering a standard or care
therapeutic to the subject in combination with the cdk1 inhibitor.
With respect to any combination treatment or therapy regimens
described herein, the cdk1 inhibitor composition can be
administered simultaneously with the other active agents, which may
be in admixture with the cdk1 inhibitor, or may be in a separate
composition. Each composition preferably includes a
pharmaceutically acceptable diluent, adjuvant, or carrier. When the
agents are separately administered, they may be administered in any
order.
[0089] In another aspect, disclosed herein is a therapeutic regimen
for treating a cancer in a mammalian subject, the method
comprising: (a) measuring phosphorylation state and/or
intracellular localization of inhibitory Apoptosis-Stimulating
Protein of p53 (iASPP) in a cancer cell from a mammalian subject,
(b) screening the cancer cell for at least one parameter selected
from the group consisting of: a p53 mutation that reduces a tumor
suppressive function of p53, an elevated measurement of mdm2
protein or mRNA, and a B-RafV600E mutation; and (c) prescribing
and/or administering a therapeutic regimen that includes an
inhibitor of iASPP phosphorylation for a subject with a cancer
characterized by elevated iASPP phosphorylation and/or iASPP
nuclear localization. In some embodiments, the therapeutic regimen
comprises prescribing and/or administering a therapeutic regimen
that includes an inhibitor of iASPP phosphorylation and an
inhibitor of mdm2-induced degradation of p53, for a subject with a
cancer characterized by elevated iASPP phosphorylation and/or iASPP
nuclear localization and the absence of a p53 mutation that reduces
the tumor suppressive function of p53. (The presence of a wild type
p53 is scored as the absence of a p53 mutation that reduces tumor
suppressive function of p53.) The therapeutic regimen optionally
comprises prescribing and/or administering a therapeutic regimen
that includes an inhibitor of iASPP phosphorylation and an
inhibitor B-RafV600E, for a subject with a cancer characterized by
elevated iASPP phosphorylation and/or iASPP nuclear localization
and the presence of the B-Raf-V600E mutation.
[0090] In some embodiments, the inhibitor of iASPP phosphorylation
is an inhibitor of cyclin B1/cdk1, such as K00616a or JNJ-7706621.
The inhibitor of mdm2-induced degradation of p53 is optionally
Nutlin3, and the inhibitor of B-RafV600E is optionally GDC-0879,
PLX-4720, Sorafenib, Tosylate Vemurafenib (also known as PLX4032,
RG7204 or RO5185426, marketed as Zelboraf.TM.).
[0091] The therapeutic regimen optionally comprises prescribing a
standard-of-care therapy to a mammalian subject in whom elevated
iASPP phosphorylation and/or elevated iASPP nuclear localization is
absent from the cancer cell.
[0092] In some variations, the invention is simply the treatment
steps without first determining the phosphorylation state of iASPP
in a cancer cell of a mammalian subject.
[0093] The invention includes use of measurement of a
phosphorylation state and/or intracellular localization of
inhibitory Apoptosis-Stimulating Protein of p53 (iASPP) in a cancer
cell from a mammalian subject for assessing the metastatic
potential of the cancer and/oe selection of a therapeutic regimen.
Such a use optionally comprises use of a measurement of at least
one parameter selected from the group consisting of: a p53 mutation
that reduces a tumor suppressive function of p53, an elevated
measurement of mdm2 protein or mRNA, and a B-RafV600E mutation; for
the selection of a therapeutic regimen for the mammalian subject.
In some embodiments, the therapeutic regimen includes an inhibitor
of iASPP phosphorylation for a subject with a cancer characterized
by elevated iASPP phosphorylation and/or iASPP nuclear
localization, and optionally wherein the therapeutic regimen
further includes an inhibitor of mdm2-induced degradation of p53,
for a subject with a cancer characterized by elevated iASPP
phosphorylation and/or iASPP nuclear localization and the absence
of a p53 mutation that reduces the tumor suppressive function of
p53, and an inhibitor B-RafV600E, for a subject with a cancer
characterized by elevated iASPP phosphorylation and/or iASPP
nuclear localization and the presence of the B-Raf-V600E
mutation.
[0094] Another aspect of the invention is a system for identifying
metastatic potential of a cancer in a human subject, the system
comprising (a) at least one processor; (b) at least one
computer-readable medium; (c) a database operatively coupled to a
computer-readable medium of the system and containing population
information correlating the phosphorylation state or intracellular
localization of iASPP in cancer and metastasis data in a population
of humans; (d) a measurement tool that receives an input about the
human subject and generates information from the input about the
iASPP phosphorylation or iASPP intracellular localization in a
cancer cell from the human subject; and (e) an analysis tool or
routine that: (i) is operatively coupled to the database and the
measurement tool, (ii) is stored on a computer-readable medium of
the system, (iii) is adapted to be executed on a processor of the
system, to compare the information about the human subject with the
population information in the susceptibility database and generate
a conclusion with respect to susceptibility to metastatic potential
of the cancer in the human subject.
[0095] In some embodiments, the database optionally includes
population information correlating the presence or absence of a p53
mutation that reduces a tumor suppressive function of p53 and
metastasis data in the population of humans, wherein the
measurement tool further generates information from the input about
the presence or absence of a p53 mutation in the cancer cell from
the human subject that reduces a tumor suppressive function of p53,
and wherein the analysis tool generates a conclusion with respect
to susceptibility to metastatic potential of the cancer in the
human subject using both information about the phosphorylation
state or intracellular localization of iASPP and the information
about the p53 mutation.
[0096] As described below in detail, some variations of a system of
the invention include a medical protocol database operatively
connected to a computer-readable medium of the system and
containing information correlating iASPP phosphorylation state (and
any of the one or more additional parameters described herein) and
medical protocols for treating human subjects with cancer. Such a
system further includes a medical protocol tool or routine to
compare or correlate the conclusion obtain from the analysis
routine and the medical protocol database, and generate a protocol
report with respect to the probability that one or more medical
protocols will achieve a therapeutic goal, or a protocol report
assessing the relative merits of different protocols for the
subject's cancer.
[0097] The invention also includes compounds, compositions,
methods, and systems for treatment of cancers.
[0098] Also provided is a composition comprising an inhibitor of
cyclin B1/cdk1-induced phosphorylation of iASPP and a
pharmaceutically acceptable carrier. The composition optionally
further comprises an inhibitor of mdm2-induced degradation of p53.
In some embodiments, the composition further comprises an inhibitor
of B-RafV600E.
[0099] Another aspect of the invention is a unit dose comprising an
inhibitor of cyclin B1/cdk1-induced phosphorylation of iASPP; an
inhibitor of mdm2-induced degradation of p53; and an inhibitor
B-RafV600E; wherein the inhibitors are packaged together, and at
least two of the inhibitors are not in admixture. In some
embodiments, the three inhibitors are not in admixture.
[0100] Yet another aspect is a method of treating a mammalian
subject with cancer, wherein the method comprises administering to
the subject an inhibitor of cyclin B1/cdk1-induced phosphorylation
of iASPP; an inhibitor of mdm2-induced degradation of p53; and an
inhibitor B-RafV600E; wherein the inhibitors are administered in
amounts effective to inhibit proliferation of the cancer. In some
embodiments, the inhibitors are optionally administered in amounts
effective to induce death of cancer cells in the mammalian
subject.
[0101] Aspects of the invention that are described herein as
methods (especially methods that involve treatment) can
alternatively be described as (medical) uses of reagents or
therapeutics. For example, in one variation, the invention is a use
of a composition that comprises a molecule that suppresses
expression or activity of cdk1 for the treatment of cancer in a
subject identified with cancer and identified with elevated
phosphorylated iASPP expression in the cancer (wherein the subject
is identified as having elevated phosphorylated iASPP expression in
the cancer by a method described herein).
[0102] In the treatment methods (or uses) described herein, the
methods optionally comprises administering a standard or care
therapeutic to the subject in combination with the inhibitor of
cyclin B1/cdk1-induced phosphorylation of iASPP; an inhibitor of
mdm2-induced degradation of p53; and/or an inhibitor B-RafV600E.
With respect to any combination treatment or therapy regimens
described herein, the cdk1 inhibitor composition can be
administered simultaneously with the other active agents, which may
be in admixture with the inhibitor of cyclin B1/cdk1-induced
phosphorylation of iASPP; an inhibitor of mdm2-induced degradation
of p53; and/or inhibitor B-RafV600E, or may be in a separate
composition. Each composition preferably includes a
pharmaceutically acceptable diluent, adjuvant, or carrier. When the
agents are separately administered, they may be administered in any
order.
[0103] The foregoing summary is not intended to define every aspect
of the invention, and additional aspects are described in other
sections, such as the Detailed Description. The entire document is
intended to be related as a unified disclosure, and it should be
understood that all combinations of features described herein are
contemplated, even if the combination of features are not found
together in the same sentence, or paragraph, or section of this
document.
[0104] In addition to the foregoing, the invention includes, as an
additional aspect, all embodiments of the invention narrower in
scope in any way than the variations defined by specific paragraphs
above. For example, certain aspects of the invention that are
described as a genus, and it should be understood that every member
of a genus is, individually, an aspect of the invention. Also,
aspects described as a genus or selecting a member of a genus,
should be understood to embrace combinations of two or more members
of the genus. Although the applicant(s) invented the full scope of
the invention described herein, the applicants do not intend to
claim subject matter described in the prior art work of others.
Therefore, in the event that statutory prior art within the scope
of a claim is brought to the attention of the applicant(s) by a
Patent Office or other entity or individual, the applicant(s)
reserve the right to exercise amendment rights under applicable
patent laws to redefine the subject matter of such a claim to
specifically exclude such statutory prior art or obvious variations
of statutory prior art from the scope of such a claim. Variations
of the invention defined by such amended claims also are intended
as aspects of the invention.
BRIEF DESCRIPTION OF THE FIGURES
[0105] FIG. 1. Nuclear iASPP is associated with the appearance of a
slow-migrating iASPP isoform that preferentially binds p53 in vivo.
A. iASPP staining and nucleus specific marker staining (Topro) in
melanoma and H1299 cells shows distinct cytoplasmic and nuclear
localization. Melanoma (WM115 and SRK-MEL37) and H1299 cells were
seeded on cover slides and immunofluorescence stained by iASPP
antibody LX49.3. Bar=20 .mu.m. B. iASPP expression pattern in a
panel of melanoma cell lines. Total cell lysate was prepared in
UREA buffer, and iASPP patterns were determined by iASPP antibody
LX49.3. C. p53 prefers to bind to the slow-migrating iASPP isoform
in melanoma. Melanoma cell lysate was prepared in NP40 buffer, and
p53 antibody (Do-1) was applied to co-immunoprecipitate iASPP (if
any) bound to p53, followed by iASPP determination by antibody
LX49.3.
[0106] FIG. 2. iASPP cellular localization and expression pattern
in various cell lines were determined by immunoassay with antibody
LX49.3. Bar=20 .mu.m. The tumor cell lines examined are indicated.
Table 1 within FIG. 2 is a summary of iASPP (slow versus fast
migrating forms) and cellular localization in the various cancer
cell lines that were examined.
[0107] FIG. 3. Mitotic arrests induces slow-migrating nuclear
iASPP. A. Mitosis-arresting (i.e., apoptosis-inducing)
chemotherapeutic drugs induce slow-migrating iASPP. H1299 cells
were treated overnight with the indicated concentrations of
mitosis-arrest drugs nocodazole, colchicine and taxol, DNA damaging
drugs, or oxidative stress inducers (1-3 h). Total lysate was
prepared in UREA buffer, and iASPP expression patterns were
determined using antibody LX49.3. B. Slow-migrating iASPP
exclusively exists in mitotic cells after drug induction.
Mitotic-arrest drugs were applied to H1299 cells to induce iASPP
mobility shift. Floating and adherent cells were collected, and
iASPP expression pattern was determined by western blotting (WB).
C. Slow-migrating iASPP exists in natural mitotic cells and
localized in nucleus (as determing by staining with the nuclear
specific marker Topro). Left panel, cells in mitosis (floating) and
interphase (adherent) were collected by mitotic shake-off, and
iASPP expression pattern was determined by WB. Right panel, H1299
cells after Taxol treatment, or mitotic shake-off were
immunofluorescence stained using antibody LX49.3. In mitotic
shake-off experiment, mitotic shake-off cells were cytospun to
slides; the remaining adherent cells after shaking were scraped off
and used as interphase control. D. Mitotic-arrest drugs induce
slow-migrating iASPP in melanomas. Melanoma cells were treated with
nocodazole overnight and iASPP expression patterns from total cell
lysate was determined by WB.
[0108] FIG. 4. A. Floating and adherent H1299 cells separately
collected by mitotic shake-off (FIG. 3C) were stained with
propidium iodide (PI), followed by FACS analysis. Left panel shows
the representative cell cycle profile of floating and adherent
H1299 cells, right panel show the calculated cell population in
various cell cycle phase as indicated. B. H1299 cells were
subjected to treatment of 10 .mu.M nocodazole for 24 hours
(Nocodazole), on ice for 30 minutes (Cold shock), or 4 mM EDTA for
1 hour (EDTA). Left panel: iASPP expression pattern was analysed by
immunoblotting. Right panel: phase contrast images were taken
before collection of the cells for WB analysis. Bar=20 .mu.m.
[0109] FIG. 5. CyclinB/cdk1 phosphorylation of iASPP at
Ser84/Ser113 that contributes to iASPP slow migration. A.
modification of iASPP is localized on N-terminus (1-478 aa). H1299
cells stably expressing V5 tagged iASPP fragments 1-478 and 482-828
were treated with nocodazole. Expression pattern of the iASPP
fragments was determined using anti-V5 antibody. B. N-terminal
iASPP (1-240) is a substrate of cyclinB/cdk1 in vitro. Beads
pre-bound with 5 .mu.g purified iASPP (1-240) (1-240, GST tag), or
iASPP (249-482) (249-482, GST tag) were incubated with 0.2 .mu.g/ml
cyclin B1/ckd1 complex in NP40 buffer (containing 1 mg/ml BSA) for
2 hours. After 4 washes, bound cyclin B1/ckd1 complex was
determined by WB. In lower panel, iASPP (1-240) and iASPP (249-482)
was mixed with cyclin B1/cdk1 complex and [.gamma.-32P]-ATP under
standard kinase assay conditions, bound [.gamma.-32P] was then
determined in SDS-PAGE. For input experiments, the duplicated
protein samples were stained by coomassie blue directly. C.
iASPP-S84AS113A cannot be phosphorylated by cyclinB1/cdk1 in vitro.
5 .mu.g iASPP (1-240) point mutants or double mutants on S84, S113,
and S120 were subjected to cyclin B1/cdk1 in vitro assay. The
average [.gamma.-32P] signal was quantified from 3 separate
experiments. D. Stably expressed iASPP-S84AS113A in IGR37 shows
reduced mobility shift. IGR37 and IGR39 cells stably expressing
iASPP mutants (tagged V5) were dissolved in UREA buffer and iASPP
expression pattern was determined by WB.
[0110] FIG. 6. Phosphorylation of iASPP on S113 during mitosis by
mass spectrometry. H1299 cells were treated with or without 10
.mu.M nocodazole for 24 h. iASPP was immunoprecipitated (from 40 mg
protein lysate) using cross-linked antibody LX49.3. Proteins were
separated by SDS-PAGE and the gel was stained with coomassie and
silvernitrate. Protein bands that are corresponding to the
molecular weights of iASPP were excised, digested with trypsin and
analysed by tandem mass spectrometry. MS/MS spectrum of the iASPP
derived tryptic peptide 100-115 of SEQ ID NO: 2 is shown, in which
b and y fragment ions are indicated. Phosphorylation is mapped to
Ser113; -98 Da: loss of phosphoric acid; ++doubly charged ion;
o:--NH2. Ser84 in derived tryptic peptide is also determined to be
phosphorylated in nocodazole treated sample; however, its status in
untreated sample remains unknown as it was not covered in generated
tryptic peptide in current assay.
[0111] FIG. 7. S84/S113 phosphorylation of iASPP prevents N- and
C-terminal self-interaction and promotes iASPP nuclear
localization. A. N- and C-terminal iASPP self-interacts, and iASPP
(1-240 of SEQ ID NO: 2)S84D/S113D fails to interact with C-terminal
iASPP in vitro. Upper panel: 10 .mu.g/ml purified GST-tagged iASPP
(1-240 of SEQ ID NO: 2) or iASPP (249-482 of SEQ ID NO: 2) were
incubated with 10 .mu.g/ml purified 6*His-tagged iASPP (625-828 of
SEQ ID NO: 2) or ASPP2 (905-1128 of SEQ ID NO: 2) in NP40 buffer
for 2 hours. iASPP (1-240 of SEQ ID NO: 2) and iASPP (249-482 of
SEQ ID NO: 2) were pulled down by glutathione beads. Bound iASPP
(625-828 of SEQ ID NO: 2) and ASPP2 (905-1128 of SEQ ID NO: 2) were
determined using anti-His antibodies. Lower panel: 10 .mu.g/ml
purified iASPP (1-240 of SEQ ID BO: 2)-GST mutants were incubated
with 10 .mu.g/ml mixture of iASPP (625-828 of SEQ ID NO: 2)-His and
ASPP2 (905-1128 of SEQ ID NO: 2)-His in NP40 buffer for 2 hours.
Interaction was determined as above. B. Modification of iASPP
abrogates N- and C-terminus self interaction and changes iASPP
molecule conformation. Upper left panel: iASPP(1-478 of SEQ ID NO:
2)-V5 was overexpressed in iASPP (-/-) MEFs and a slow-migrating
expression pattern was induced by nocodazole treatment. Cells were
suspended in NP40 buffer, and interactions between overexpressed
iASPP(1-478 of SEQ ID NO: 2)-V5 and purified iASPP(625-828 of SEQ
ID NO: 2)-His were determined by co-immunoprecipitation. Cartoon
figure shows the scheme of selectively binding between unmodified
iASPP(1-478 of SEQ ID NO: 2) and iASPP(625-828 of SEQ ID NO: 2).
Lower panel: endogenous iASPP in H1299 showed a slow-migrating
pattern of expression after nocodazole treatment. Cells were
suspended in NP40 buffer, and interaction between endogenous iASPP
and iASPP (1-240 of SEQ ID NO: 2)-GST was determined by
co-immunoprecipitation. Cartoon figure shows the scheme of
selective binding between modified iASPP and iASPP(1-240 of SEQ ID
NO: 2). Upper right panel: H1299 cells were treated with or without
nocodazole and iASPP mobility shift were determined in
non-denaturing or denaturing WB. C. N-terminal iASPP binds N813 and
Y814 of C-terminal iASPP (SEQ ID NO: 2). 20 .mu.l iASPP (1-240 of
SEQ ID NO: 2)-GST pre-bound glutathione beads were incubated with 3
.mu.l in vitro translated iASPP (625-828 of SEQ ID NO: 2)-V5
mutants in NP40 buffer. After 4 washes, the interaction between
them was determined by co-immunoprecipitation using anti-GST and
anti-V5 antibodies. D. N-terminal iASPP prevents nuclear
translocation of C-terminal iASPP in vitro. Purified iASPP (625-828
of SEQ ID NO: 2) was labeled with FITC and incubated with digitonin
treated H1299 cells in presence of 0-1 mg/ml iASPP (1-240 of SEQ ID
NO: 2). Nuclear iASPP (625-828 of SEQ ID NO: 2)-FITC was determined
by direct FITC fluorescence. iASPP(1-240)S84D/S113D and
ASPP2(905-1128 of SEQ ID NO: 2) were used as control. Parallel
experiments were done for all protein samples, and representative
images were shown. Bar=20 .mu.m. Lower bar graph shows the
percentage of FITC positive nucleus after treatment from 3
experiments. >100 cells were counted. E. iASPP S84D/S113D shows
stronger nuclear staining than wild type iASPP in iASPP(-/-) MEFs.
iASPP-V5 mutants were stably expressed in iASPP(-/-) MEFs; cellular
localization was determined by V5 antibody. Bar=20 .mu.m.
[0112] FIG. 8. N-terminal iASPP binds N813 and Y814 of C-terminal
iASPP. The bar graph (according to FIG. 4C in manuscript) shows the
calculated binding efficiency between iASPP (1-240)-GST and iASPP
(625-828)-V5 mutants from two individual experiments. Binding
efficiency=bound iASPP (625-828) signal/input iASPP (625-828)
signal. The upper cartoon figure (modified from Robinson's
publication) shows the residues (in red) involved in iASPP
self-interaction according to the current assay; the percentages
under residues represent the remaining bind signal after Alanine
point mutation; the grey area represents the putative p53 binding
space of iASPP, according to our recently determined iASPP
(625-828) structure (Robinson et al., 2008).
[0113] FIG. 9. Phospho-mimic iASPP, iASPP(S84D/S113D), is more
potent than wild type iASPP in binding and inhibiting p53. A. iASPP
S84D/S113D binds p53 better than WT iASPP in vitro. in vitro
translated iASPP-V5 (WT) or its mutant iASPP(S84D/S113D)
(S84D/S113D) (Input) were incubated with nickel beads that were
pre-bound with either p53(2-193)-His (p53) or BSA (BSA) to carry
out an immunoprecipitation assay (IP). The amount of iASPP that was
co-immunoprecipitated with p53 was detected by an anti-V5 antibody
(iASPP V5 panel) whereas the amount of p53 immunoprecipitated was
detected by an anti-His antibody (p53(2-193)-His panel). B iASPP
S84D/S113D binds p53 better than WT iASPP in transfected cells.
iASPP-V5 (WT) or its mutant iASPP(S84D/S113D) (S84D/S113D) and p53
were co-transfected in H1299 cells (Input). Cell lysate was
prepared in NP40 buffer; p53 antibody (Do-1) was applied to
co-immunoprecipitate co-transfected iASPP and its mutant. The
amount of p53 pulled down is shown in (p53 panel) whereas p53 bound
iASPP was determined by anti-V5 antibody (iASPP V5 panel). IgG was
used as a negative control. C iASPP S84D/S113D inhibits p53
reporter activity better than wild type iASPP. The bar graph shows
the effect of iASPP (WT) or its mutant, iASPP(S84D/S113D) (DD) on
the transcriptional activity of p53 on PIG3 in Saos-2 cells. Values
are the mean.+-.SD of three independent experiments. Lower panel:
the expression levels of various transfected proteins as indicated.
Tubulin is used as a loading control. D iASPP S84D/S113D confers
iASPP(-/-) MEFs higher resistance to apoptosis than wild type
iASPP. Ras and E1A transformed iASPP(-/-) MEFs (Refers as
iASPP(-/-)MEFS) stably expressing vector (Vec), iASPP(WT), or iASPP
S84D/S113D (84D/113D) were subjected to various treatment as
indicated for 24 hours. Cells were dissolved in urea buffer. The
expression levels of known apoptosis markers were detected in
immunoblotting using D214 or D175 to detect cleaved PARP (c-PARP)
or caspase 3 (c-caspase 3) respectively. E. iASPP S84D/S113D
confers upon MEFs more resistance to Chemotherapeutic drug induced
apoptosis. iASPP knockout MEFs stably expressing vector, iASPP, or
iASPP (S84D/S113D) were subjected to apoptosis induced drugs for 24
hours. Cells were dissolved in urea buffer and apoptosis markers
(cleaved PARP, caspase-3) were determined by WB.
[0114] FIG. 10. Ser84/Ser113-phosphorylated nuclear iASPP is
enriched in wild type p53 expressing metastatic tumor. FIG. 10A.
Nuclear iASPP shows association with metastasis in melanoma tissue.
Left panel: representative iASPP staining pattern in primary
tumours (Mdn40.0) and lymph node metastases (Mdn70.0) in melanoma
tissue microarray cores. Bar=50 .mu.m. Tumor tissues were stained
with LX49.3. Right panel: the overall nuclear iASPP level is
significantly higher in lymph node metastases compared to primary
tumours in 135 melanoma tissue microarray cores (U=1740.000,
r=-0.21, p<0.01). iASPP expression level=Intensity of staining
(0-none, 1-weak, 2-moderate, 3-strong).times.Proportion of cells
exhibiting staining (0-100% in 5-10% increments). Results were
analysed using the Mann-Whitney test (U). The effect size is
denoted by r=z/ n(>0.3=medium effect, >0.5=large effect). **
p<0.01. FIG. 10B. Slow-migrating nuclear iASPP lost epitope of
LX128.5. The iASPP immunostaining pattern in a panel of melanoma
cells were determined by LX128.5 antibody (immunostaining pattern
for LX49.3 was shown in FIG. 10A). Bar=10 .mu.m. The iASPP
expressing pattern in melanoma cells (total cell lysate in urea
buffer) was determined by LX128.5 and LX49.3 antibody,
respectively. FIG. 10C. p53 wild type melanomas show high ratio of
slow-migrating iASPP, and slow-migrating iASPP level shows positive
correlation with cyclin B1 level. Left panel: cell lysate from 18
melanoma cell lines was prepared in urea buffer and indicated
proteins were determined by WB. Samples with wild type p53 and
overexpressed MDM2/MDMX are labelled with stars and triangles,
respectively. Right panel: bar graph shows the iASPP modified ratio
and p53 status (indicated on the bar). Lower table: wild type p53,
overexpression of MDM2, and overexpression of MDMX are shown in
melanomas. In FIG. 10C, the arrow head shows the position of the
slow-migrating iASPP isoform while the arrow shows the
fast-migrating isoform. FIG. 10D. iASPP expression pattern in
paired primary/metastatic cell lines. Paired human tumor cell line.
IGR39 (primary) IGR37 (metastatic) LNCaP (primary) LNCaP3
(metastatic). Level of slow-migrating nuclear iASPP correlates with
metastasis. Red (top) arrow heads=slow migrating modified iASPP.
Black (bottom) arrow heads=fast migrating unmodified iASPP.
[0115] FIG. 11. FIG. 11A. Panels of iASPP antibodies lose epitopes
in slow-migrating iASPP and iASPP S84D/S113D. Upper cartoon figure:
epitope locations of iASPP antibodies. H1299 lysate panel: H1299
cells were treated with nocodazole overnight, and cell lysate in
urea buffer was probed using panels of iASPP antibodies as
indicated by immunoblotting. iASPP IVT panel: in vitro translated
wild type iASPP and iASPP S84D/S113D were probed using panels of
iASPP antibodies by immunoblotting. Melanomas panel: melanoma
lysate in urea buffer was probed with iASPP antibody LX49.3 by
Western blot (WB). After stripping, the membrane was re-probed with
LX128.5. FIG. 11B. Cyclin B1 levels show association with
slow-migrating iASPP levels in 18 melanoma cell lines. Upper panel:
bar graph shows the relative levels of modified iASPP (bar on left
side of each lane) and cyclin B1 (bar on right side of each lane)
in 18 melanoma cell lines. FIG. 11C. iASPP modification show
positive correlation with cyclin B1 level and p53 status in
prostate cancer cells; Left panel; Cell lysate from panel of
prostate cancer cell lines was prepared in UREA buffer and
indicated proteins were determined by WB. Right bar graph: the
association between iASPP modification and cyclin B1 level/p53
status.
[0116] FIG. 12. Restoring the tumour suppressive function of p53
and inhibiting BRAFV600E causes synthetic suppression of melanoma
cells. FIG. 12A. H1299 cells were co-treated with 10 .mu.M
nocodazole and indicated inhibitors against mitosis related kinase
overnight. Total cells were collected and lysed in urea buffer and
iASPP express pattern was determined by immunoblotting. FIG. 12B.
Blocking of iASPP modification by K00616a increases apoptosis
sensitivity of melanomas to Nutlin3. Cells were transfected with
siRNA as shown and pre-seeded on 6 well plates ON, 0.3 .mu.M cyclin
B1/cdk1 inhibitors K00616a were then added for 48 hours to block
iASPP modification. Cells were then treated with 0.3 .mu.M K00616a,
20 .mu.M nutlin3, or combinations of these drugs for another 72
hours. Cells were collected and cell cycle status, growth
potential, and protein expression pattern were then determined by
PI staining/FACS analysis, colony formation assay, and WB,
respectively. The upper bar graph shows the averaged sub-G1 ratio
after drug treatment from 3 separated experiments; the lower bar
graph shows the averaged cell colony number by seeding treated
cells for 1-2 weeks. FIG. 12C. Restoring of p53 function by K00616a
and nutlin3 increase apoptosis sensitivity of melanomas to PLX4032.
Cells were pre-seeded on 6 well plates for overnight, 0.3 .mu.M
cyclin B1/cdk1 inhibitors K00616a were then added for 48 hours to
block iASPP modification. Cells were then treated with 0.3 .mu.M
K00616a, 20 .mu.M nutlin3, 1 .mu.M PLX4032, or combinations of
these drug for another 72 hours. Cells were collected and cell
cycle status, growth potential, and protein expression pattern were
then determined by PI staining/FACS analysis, colony formation
assay, and WB, respectively. The upper bar graph shows the averaged
sub-G1 ratio after drug treatment from 3 separate experiments; the
lower bar graph shows the averaged cell colony number by seeding
treated cells for 1-2 weeks.
[0117] FIG. 13. FIG. 13A. H1299 cells were co-treated with 10 .mu.M
nocodazole and indicated inhibitors against mitosis related kinase
overnight. Total cells were collected and lysed in urea buffer and
iASPP express pattern was determined by WB. The table shows
information of inhibitors. (+)=iASPP modification is blocked
(-)=iASPP modification is not blocked; (.+-.)=iASPP modification is
partially blocked. Inhibitors were supplied by Structural Genomics
Consortium (Fedorov et al., 2007). FIG. 13B. Cellular population
and protein expression patterns in melanoma cells after treatments
indicated in FIG. 13A. Bar graph shows the corresponding cell
population in different phases of the cell cycle. SubG1 is the
uppermost portion of the bars, being barely visible in lanes 1 in
the first two bar graphs and is also barely visible in lanes 1-7 of
the third bar graph. Lower panel shows iASPP and p53 expression
patterns after indicated treatments.
[0118] FIG. 14. S84 and 5113 phosphorylation increase potential of
iASPP in inhibiting p53. Normal: C-terminus of iASPP (625-828)
binds N-terminus (1-240), forming iASPP dimer (or self-folded iASPP
monomer). The key C-terminal nuclear localization signal (NLS,
located in Ankyrin repeat domain) and p53 binding epitope
(involving N813) are masked, leading to cytoplasmic localization of
iASPP. Mitosis/melanoma: with cellular overexpression of cyclin
B1/cdk1 (during mitosis or in some melanomas), N-terminal Ser84 and
S113 are phosphorylated. This disrupts iASPP self-interaction,
exposing the C-terminal NLS and p53 binding epitope. The exposed
C-terminal NLS drives phosphorylated iASPP into the nucleus, where
it binds to and inhibits p53. Synthetic inhibition: restoration of
p53 by blocking iASPP and MDM2 increases the apoptosis sensitivity
of melanoma cells that express wild type p53; blocking of the B-RAF
pathway by PLX4032 suppresses the proliferation of melanoma hosting
B-RAF V600E. Reactivation of p53-induced apoptosis with concurrent
inhibition of the B-RAF proliferation pathway induces synthetic
inhibition in melanomas.
[0119] FIG. 15 is an exemplary system comprising exemplary computer
components.
[0120] FIG. 16 is a flow chart depicting system components and
operation.
[0121] FIG. 17 is another flow chart depicting system components
and operation.
[0122] FIG. 18. S84/S113 phosphorylated nuclear iASPP is enriched
in wild type p53 expressing melanoma cells. FIG. 18A shows
representative iASPP and cyclin B1 staining patterns in 142 human
melanoma tissue microarray cores (left panel). Bar=50 .mu.m. Tumors
expressing strong cyclin B1 showed significantly higher nuclear
iASPP levels (U=340.500, r=-0.28, p<0.05) (right panel). FIG.
18B shows that lymph node metastatic tumors ("metastasis" right
panel) demonstrate greater occurrence of high cyclin B1 and high
nuclear iASPP expression compared to primary tumors ("primary" left
panel). The bars provided in the "primary" panel from left to right
are as follows: low iASPP/low cyclin B1; high iASPP/low cyclin B1;
low iASPP/medium cyclin B1; high iASPP/medium cyclin B1; low
iASPP/high cyclin B1 and high iASPP/high cyclin B1. The bars
provided in the "metastasis" panel from left to right are as
follows: low iASPP/low cyclin B1; high iASPP/low cyclin B1; low
iASPP/medium cyclin B1; high iASPP/medium cyclin B1; low iASPP/high
cyclin B1 and high iASPP/high cyclin B1. A survival curve shows
that high nuclear iASPP correlates with poor survival in all of
three groups of cyclin B, i.e., groups expressing low, medium, and
high cyclin B1 (p=0.033).
[0123] FIG. 19. Restoring p53 by inhibiting mdm2 and iASPP
suppresses melanoma growth in vitro and in vivo. FIG. 19A. Upper
left panel: H1299 cells were treated with control (Ctr) or cyclin
B1 RNAi to knock down cyclin B1 in the presence (+) or absence (-)
of 10 .mu.M nocodazole. Upper right panel: double
immunofluorescence staining shows colocalization of nuclear cyclin
B1/ASPP, but not cdk1/iASPP. H1299 cells expressing transfected
cyclinB1 or cdk1 are labeled by white arrows. Bar=20 .mu.m. Lower
left panel: H1299 cells were co-treated with 10 .mu.M nocodazole
and various mitosis related kinase inhibitors overnight. Lower
right panel: WM278 cells were treated with DMSO, 0.4 .mu.M
JNJ-7706621 (an inhibitor of cyclin B1/CDK1, also referred to
herein as"JNJ"), or 10 .mu.M nocodazole overnight, and were lysated
and incubated with (+) or without (-) calf intestinal phosphatase
(CIP) for 1 hour, followed by p53 co-immunoprecipitation. Arrows
and arrow heads mark the positions of the fast or slow-migrating
iASPP isoforms respectively. FIG. 19B. WM278 cells were pre-treated
with 0.4 .mu.M JNJ for 48 hours to block iASPP modification. Cells
were then treated with 0.4 .mu.M JNJ, 20 .mu.M Nutlin3 or a
combination as indicated for another 72 hours in the presence or
absence of control, iASPP, and p53 RNAi as indicated. The left bar
graph shows the percentage of Annexin V positive cells and the
right bar graph shows the percentage of treated cells that formed
colonies. Bars (from left to right) in both bar graphs correspond
to cells treated with DMSO, JNJ, Nut and JNJ+Nut, respectively. The
number of colonies formed in DMSO treated cells were set as 100%.
The bar graphs (mean.+-.SD) were derived from three independent
experiments. *p<0.05. FIG. 19C. In vitro treatment of
JNJ+Nutlin3, as described herein above, suppresses B16 melanoma
cell growth in vitro (left panel, colony assay) and in vivo (right
panel, xenograft model). Bar graphs are shown as mean.+-.SD
(*p<0.05, n=4 for each group). Lower panel shows representative
dishes and isolated tumors, respectively. FIG. 19D. Untreated B16
cells were subcutaneously injected into flanks of mice at day 1. At
day 7, tumors were visible, and compounds (i.e., JNJ, Nutlin3,
JNJ+Nutlin3, and control vehicle (DMSO)) were intraperitoneally
(i.p) injected. Compound administration and tumor size measurements
were repeated every two days from day 7 onwards. Bar graphs are
shown as mean.+-.SD (*p<0.05, n=4). Lower panels show
representative image of isolated tumors. FIG. 19E. Representative
immunofluorescence staining of iASPP and p53 in tumor section
derived from FIG. 19D. White dotted circles show the representative
nuclear rim derived from Topro staining. Bar=20 .mu.m. (C-D),
treated or untreated B16 cells (1.times.10.sup.6) were
subcutaneously injected into C57BL/6 mice at day 1 and tumor sizes
were measured every two days. Tumors were weighted at the end-point
(day 13).
[0124] FIG. 20. Restoring p53 by inhibiting mdm2 and iASPP
suppresses melanoma growth in vitro and in vivo. FIG. 20A. H1299
cells were treated with DMSO or 10 .mu.M nocodazole in the presence
or absence of 2 or 10 .mu.M of mitosis-related kinase inhibitors as
indicated. Table 2 within FIG. 20A shows the efficacy of mitotic
kinase inhibitors to reduce iASPP modification, which ranged from
very effective (+++), to effective (++), and to weakly effective
(+). Those which failed to affect iASPP mobility shift are labeled
as negative (-). FIG. 20B. Left panel: representative p53 level
after Nutlin3 treatment for 24 hours in a variety of cells. Right
bar graphs show the sub-G1 proportion of WM278 after treatment as
that described in FIG. 19B (in upper panel, 10 .mu.M and 20 .mu.M
Nutlin3 were used in parallel for comparison). From left to right
of the upper right bar graph of FIG. 20B, the bars correspond to
DMSO, Nut and JNJ+Nut, respectively. From left to right of the
bottom bar graph of FIG. 20B, the bars correspond to DMSO, JNJ, Nut
and JNJ+Nut, respectively. FIG. 20C. Me300, SK-MEL37, and B16 cells
were treated as above (For B16 panel, 10 .mu.M Nutlin3 was used)
and sub-G1 proportion was analyzed. In (B) and (C), the bar graphs
(mean.+-.SD) were derived from three independent experiments.
*p<0.05. FIG. 20D. Left panel: untreated B16 cells were
subcutaneously injected into mice at day 1. At day 3, cells settled
down and JNJ, Nutlin3, JNJ+Nutlin3, and control vehicle (DMSO) were
intraperitoneally (i.p) injected. Compound administration and tumor
size measurement were repeated every two days from day 7 onwards.
Bar graphs are shown as mean.+-.SD (*p<0.05, n=6). Lower panels
show representative images of isolated tumors. Right panel:
representative H & E staining in B16 tumor section. Bar=20
.mu.m.
[0125] FIG. 21. Restoring apoptotic function of p53 cooperates with
BRAFV600E inhibition to suppress human melanoma cell growth. WM278
cells were treated with 0.4 .mu.M JNJ, 20 .mu.M Nutlin3, 1 .mu.M
PLX4032, or combinations as indicated in the presence or absence of
control, iASPP or p53 RNAi as indicated. The upper bar graph (FIG.
21A) shows the percentage of Annexin V positive cells and the lower
bar graph (FIG. 21B) shows the percentage of treated cells that
formed colonies. The bar graphs (mean.+-.SD) were derived from
three independent experiments. *p<0.05. Lower images show
representative Annexin V/7-AAD profile and dishes, respectively.
FIG. 21C. Diagrams showing how cyclinB1/cdk1 phosphorylation
controls iASPP localization in interphase (interphase) or mitosis
or tumor cells (mitosis/melanomas). Blue and red stars (*) indicate
the C- and N-terminal contact residues and the phosphorylation
status is labeled as (P). The bottom diagram summarizes the
pathways that PLX4032, Nutlin3 and JNJ used to suppress melanoma
cell growth.
[0126] FIG. 22. Restoring the apoptotic function of p53 cooperates
with BRAFV600E inhibition to suppress human melanoma cell growth.
FIG. 22A. Bar graph shows the sub-G1 proportion of WM278 after
treatment as described in FIG. 21A. 1 .mu.M and 5 .mu.M PLX4032
were used in parallel for comparison. FIG. 22B. WM278 cells were
treated as described for FIG. 21A. From left to right, the bars
correspond to DMSO, JNJ, Nut and JNJ+Nut, respectively. The bar
shows the percentage of Sub-G1 cells. The bar graphs (mean.+-.SD)
were derived from three independent experiments. *p<0.05. C.
Me300, SK-MEL37, and B16 cells were treated as described herein and
sub-G1 cells were analyzed as described herein.
[0127] FIG. 23A provides the sequence of the iASPP peptide (amino
acids 107-118 of SEQ ID NO: 2) used to generate the anti-iASPP
phosphoserine 113 antibody as described in Example 1.
[0128] FIG. 23B is a gel showing that antibody binds
phosphor-specific ser113 iASPP.
[0129] FIG. 23C is a graph showing the specific binding the
anti-iASPP ser113 phosphospecific antibody to phosphorylated
iASPP.
DETAILED DESCRIPTION
[0130] The ASPP family (Apoptotic-Stimulating Proteins of p53) is
newly identified as a family of p53 interacting proteins which
includes members that may contribute to site selectivity by
enhancing the activation of apoptotic rather than growth arrest
target genes. This finding indicates that the ASPP family
cooperates with p53, and plays an important role in controlling the
decision between life and death.
[0131] Three members, ASPP1, ASPP2 and iASPP have been identified
in the family (Bergamaschi et al., 2004; Bergamaschi et al., 2003).
All members contain ankyrin repeats, an SH3 domain and a
proline-rich domain in their carboxyl terminus. This conserved
C-terminus sequence involves the most important binding sites with
the partners identified so far (Trigiante and Lu, 2006). The N
terminus of iASPP has no homology with ASPP1 and ASPP2. The
sequence similarities among the ASPP family members indicate that
ASPP1 and ASPP2 probably have similar biological functions that
differ from that of iASPP. Indeed, existing studies have
demonstrated that ASPP1 and ASPP2 are specific pro-apoptotic
regulators of p53 (Samuels-Lev et al., 2001); while iASPP is an
inhibitor and antagonizes ASPP1 and ASPP2 function (Bergamaschi et
al., 2003). Importantly, iASPP is the only member existing in C.
elegans, which indicates that iASPP is an evolutionarily conserved
inhibitor of p53.
[0132] It was recently reported that iASPP inhibits cellular
senescence in vitro and epithelium stratification in vivo (Notari
et al., 2011). iASPP is predominantly expressed as nuclear protein
in basal epithelium that also co-expresses p63, a proliferative
compartment of epidermis. iASPP migrates to the cytoplasm in
differentiated epithelial cells, and cellular location of iASPP
must therefore play a crucial role in controlling its function upon
epithelium differentiation. Since many of the proteins with which
iASPP interacts are transcription factors, including p53, p63 and
p65Nfkb, it is likely that nuclear iASPP is more active than
cytoplasmic iASPP in regulating transcription.
[0133] Previous studies have shown that the C-termini of ASPP2 and
iASPP have intrinsic nuclear localization properties (Slee et al.,
2004). It has also been postulated in the past that ankyrin repeat
domains (ARDs) may represent a unique type of nuclear localization
signal similar as that shown for I.kappa.B.alpha. (Sachdev et al.,
1998).
[0134] The present application is based, in part, on the discovery
that phosphorylation of iASPP at one or more serine residues in the
human iASPP amino acid sequence and/or intracellular localization
of iASPP in a cancer cell correlates with, and is believed to have
an effect on the metastatic potential of a cancer cell. Identifying
that iASPP is regulated by serine phosphorylation and the
associated downstream event is not only valuable for understanding
iASPP regulated p53-dependent or -independent cellular signaling
pathways but also is useful to select more effective treatment
regimens and/or develop new strategies of cancer treatment as
described herein.
[0135] I. Diagnostic Methods
[0136] In one aspect, described herein is a method of evaluating
metastatic potential of a cancer comprising measuring
phosphorylation state and/or intracellular localization of iASPP in
a cancer cell from a mammalian subject and diagnosing metastatic
potential of the cancer based on the phosphorylation state and/or
intracellular localization of iASPP in the cancer cell measured
according to the measuring step. Such classification of tumors is
useful for treating human subjects in a clinical setting. Such
classification also is useful for laboratory research involving
experimental animals, e.g., genetically engineered mouse models of
cancer. Such methods also could be described as methods of
stratifying or grading tumors into, e.g., two, three, four, five or
more categories based on this measurement.
[0137] The invention is contemplated to be useful with respect to
any cancer type. Exemplary cancers include, but are not limited to,
adrenocortical carcinoma, AIDS-related cancers, AIDS-related
lymphoma, anal cancer, anorectal cancer, cancer of the anal canal,
appendix cancer, childhood cerebellar astrocytoma, childhood
cerebral astrocytoma, basal cell carcinoma, skin cancer
(non-melanoma), biliary cancer, extrahepatic bile duct cancer,
intrahepatic bile duct cancer, bladder cancer, uringary bladder
cancer, bone and joint cancer, osteosarcoma and malignant fibrous
histiocytoma, brain cancer, brain tumor, brain stem glioma,
cerebellar astrocytoma, cerebral astrocytoma/malignant glioma,
ependymoma, medulloblastoma, supratentorial primitive
neuroectodeimal tumors, visual pathway and hypothalamic glioma,
breast cancer, bronchial adenomas/carcinoids, carcinoid tumor,
gastrointestinal, nervous system cancer, nervous system lymphoma,
central nervous system cancer, central nervous system lymphoma,
cervical cancer, childhood cancers, chronic lymphocytic leukemia,
chronic myelogenous leukemia, chronic myeloproliferative disorders,
colon cancer, colorectal cancer, cutaneous T-cell lymphoma,
lymphoid neoplasm, mycosis fungoides, Seziary Syndrome, endometrial
cancer, esophageal cancer, extracranial germ cell tumor,
extragonadal germ cell tumor, extrahepatic bile duct cancer, eye
cancer, intraocular melanoma, retinoblastoma, gallbladder cancer,
gastric (stomach) cancer, gastrointestinal carcinoid tumor,
gastrointestinal stromal tumor (GIST), germ cell tumor, ovarian
germ cell tumor, gestational trophoblastic tumor glioma, head and
neck cancer, hepatocellular (liver) cancer, Hodgkin lymphoma,
hypopharyngeal cancer, intraocular melanoma, ocular cancer, islet
cell tumors (endocrine pancreas), Kaposi Sarcoma, kidney cancer,
renal cancer, kidney cancer, laryngeal cancer, acute lymphoblastic
leukemia, acute myeloid leukemia, chronic lymphocytic leukemia,
chronic myelogenous leukemia, hairy cell leukemia, lip and oral
cavity cancer, liver cancer, lung cancer, non-small cell lung
cancer, small cell lung cancer, AIDS-related lymphoma, non-Hodgkin
lymphoma, primary central nervous system lymphoma, Waldenstram
macroglobulinemia, medulloblastoma, melanoma, intraocular (eye)
melanoma, merkel cell carcinoma, mesothelioma malignant,
mesothelioma, metastatic squamous neck cancer, mouth cancer, cancer
of the tongue, multiple endocrine neoplasia syndrome, mycosis
fungoides, myelodysplastic syndromes,
myelodysplastic/myeloproliferative diseases, chronic myelogenous
leukemia, acute myeloid leukemia, multiple myeloma, chronic
myeloproliferative disorders, nasopharyngeal cancer, neuroblastoma,
oral cancer, oral cavity cancer, oropharyngeal cancer, ovarian
cancer, ovarian epithelial cancer, ovarian low malignant potential
tumor, pancreatic cancer, islet cell pancreatic cancer, paranasal
sinus and nasal cavity cancer, parathyroid cancer, penile cancer,
pharyngeal cancer, pheochromocytoma, pineoblastoma and
supratentorial primitive neuroectodermal tumors, pituitary tumor,
plasma cell neoplasm/multiple myeloma, pleuropulmonary blastoma,
prostate cancer, rectal cancer, renal pelvis and ureter,
transitional cell cancer, retinoblastoma, rhabdomyosarcoma,
salivary gland cancer, ewing family of sarcoma tumors, Kaposi
Sarcoma, soft tissue sarcoma, uterine cancer, uterine sarcoma, skin
cancer (non-melanoma), skin cancer (melanoma), merkel cell skin
carcinoma, small intestine cancer, soft tissue sarcoma, squamous
cell carcinoma, stomach (gastric) cancer, supratentorial primitive
neuroectodermal tumors, testicular cancer, throat cancer, thymoma,
thymoma and thymic carcinoma, thyroid cancer, transitional cell
cancer of the renal pelvis and ureter and other urinary organs,
gestational trophoblastic tumor, urethral cancer, endometrial
uterine cancer, uterine sarcoma, uterine corpus cancer, vaginal
cancer, vulvar cancer, and Wilm's Tumor. In some embodiments, the
cancer cell is from a cancer selected from the group consisting of
melanoma, prostate cancer, head cancer and neck cancer. In some
embodiments, the cancer cell is from a primary tumor from the
subject.
[0138] In some embodiments, the method further comprises screening
the cancer cell for at least one parameter selected from the group
consisting of: a p53 mutation that reduces a tumor suppressive
function of p53, an elevated measurement of mdm2 protein or mRNA,
and a B-RafV600E mutation; and diagnosing metastatic potential of
the cancer based on the phosphorylation state and/or intracellular
localization of iASPP in the cancer cell and from the presence or
absence of the at least one parameter. The at least one parameter
optionally comprises the p53 mutation that reduces a tumor
suppressive function of p53. More than 26,000 somatic mutation data
of p53 has been collected and appear in the international agency
for research on cancer (IARC) TP53 database version R14
(www-p53.iarc.fr/). The absence of such a p53 mutation can be
characterized as the presence of a wild type functioning p53.
[0139] In some variations, to provide a basis for the
identification of cancers at risk for metastasis, phosphorylated
iASPP measurements (or intracellular localization measurements)
from multiple human subjects are obtained, both in healthy tissues
and cancer tissue, to establish a data set of phosphorylated iASPP
expression. With an established data set, a variety of standard
statistical analyses can be performed to identify when a
measurement of phosphorylated iASPP is elevated, e.g., in a
statistically significant manner relative to a healthy control. The
data set can be used to stratify cancers into two, three, four,
five or more categories. For example, in some embodiments, the
determination that 0%-20% of the iASPP in the sample is
phosphorylation is indicative that the cells in the sample are not
cancerous. A determination that about 20% to about 50% of the iASPP
in the sample is phosphorylated is indicative that the cells in the
sample are from a primary tumor (i.e., a tumor that has not yet
metastasized). A determination that about 50% to about 80% of the
iASPP in the sample is phosphorylated is indicative that the cells
in the sample are from a primary tumor in transition to metastasis.
A determination that about 80% to about 100% of the iASPP in the
sample is phosphorylated is indicative that the cells in the sample
are from a metastasized tumor.
[0140] To assess the relative level of phosphorylated iASPP
expression, the level of phosphorylated iASPP expression in a
cancer tissue sample is subjected to one or more of various
comparisons. In general, it can be compared to: (a) phosphorylated
iASPP expression level(s) in normal tissue from the organ in which
the cancer originated; (b) phosphorylated iASPP expression level(s)
in a collection of comparable cancer tissue samples; (c)
phosphorylated iASPP expression level(s) in a collection of normal
tissue samples; or (d) phosphorylated iASPP expression level(s) in
an arbitrary standard. In some embodiments, a phosphorylated iASPP
measurement of at least 1.0, 1.5, 2.0, 2.5 or at least 3.0 standard
deviation(s) greater than a median phosphorylated iASPP measurement
in corresponding healthy tissue is scored as elevated
phosphorylated iASPP expression. In other embodiments, a
phosphorylated iASPP measurement that is statistically
significantly greater than phosphorylated iASPP measurements in
corresponding healthy tissue, with a p-value less than 0.1, or less
than 0.05, or less than 0.01, or less than 0.005, or less than
0.001 is scored as elevated phosphorylated iASPP expression. As a
data set enlarges, the comparison can be refined by stratifying the
data for additional variables, such as the age, sex, ethnicity,
body mass, smoking habits or other factors that differentiate
subjects.
[0141] In some embodiments, the screening methods described herein
comprise comparing a measurement of phosphorylated iASPP (or iASPP
localization) in the cancer tissue sample with phosphorylated iASPP
(ir iASPP localization) measurement in healthy tissue of the same
type as the tumor or increased nuclear localization, wherein
elevated phosphorylated iASPP expression in the tumor compared to
the healthy tissue, identifies the cancer as being at greater risk
for metastasis.
[0142] In some embodiments, the measuring comprises measuring the
phosphorylation state and/or intracellular localization of iASPP in
a plurality of cancer cells from the mammalian subject and
diagnosing metastatic potential of the cancer based on an average
measurement from the plurality of the cells, or from a percentage
of the cells that exceed a cut-off or reference measurement.
[0143] The diagnosing, in some embodiments, is made from the
percentage of total iASPP that is phosphorylated or the percentage
of total iASPP that is localized in the nucleus of the cell.
[0144] The level of phosphorylated iASPP in a sample, in some
embodiments, can be compared to a predetermined "cut-off"
concentration of phosphorylated iASPP that has been determined from
observations to represent an elevated measure of phosphorylated
iASPP that (when equaled or exceeded) is predictive of metastatic
potential of a cancer. Determination of a suitable cut-off is made
using, e.g., statistical analysis of phosphorylated iASPP
concentration data collected from multiple healthy and/or cancer
patients. If a "cut-off" value is employed, the cut-off
concentration preferably is statistically determined to have
optimal discriminating value to identify those cancers with
elevated metastatic potential It will be appreciated that
statistical analysis of a dataset will permit clinicians to make
informed decisions based on concentrations other than the optimal
discriminating concentration (e.g., above or below the optimal
discriminating concentration). For example, using
receiver-operating-characteristic curves, or using other
statistical summaries of phosphorylated iASPP concentration and
observed metastases according to the invention, the practitioner is
capable of selecting a cut-off phosphorylated iASPP concentration
having a desired level of sensitivity or specificity for predicting
metastatic potential of a cancer. Considerations regarding the
probability of success of targeted therapy based on the
phosphorylated iASPP concentration, versus the probability of
success of alternative therapies based on any available clinical
data, can guide the selection of an appropriate cut-off
concentration of phosphorylated iASPP for making a treatment
decision.
[0145] In some embodiments, the cut-off is determined by measuring
total iASPP in the sample, and determining an amount of
phosphorylated iASPP in the total iASPP, wherein the determination
that at least about 50% (or at least about 60%, or at least about
70%, or at least about 80%, or at least about 90% or at least about
95%) or more of the total iASPP in the sample is phosphorylated at
one or both of Ser84 and Ser113 indicates that the cancer has an
increased risk for metastasis.
[0146] The cut-off could also be determined by identifying a ratio
of phosphorylated iASPP to total iASPP within a cell, wherein an
increased amount of phosphorylated iASPP compared to the amount of
total iASPP in the cell indicates that the cancer has a increased
risk for metastasis.
[0147] As described elsewhere herein, the screening methods
described herein may optionally comprise the step of prescribing
for or administering to the subject identified as having elevated
phosphorylated iASPP expression in the biological sample a
composition comprising a molecule that suppresses expression or
activity of cdk1, a molecule that inhibits iASPP phosphorylation,
an inhibitor of mdm2-induced degradation of p53 and/or an inhibitor
of B-RafV600E. By "prescribing" is meant providing an order or
authorization for the therapy, which may be dispensed to the
subject for self-administration and/or administered by a medical
professional that is different from the prescribing
professional.
[0148] A. Methods of Measuring iASPP Phosphorylation in a
Sample
[0149] The terms evaluation of measurement of "phosphorylation
state of iASPP" and "iASPP phosphorylation" as used herein refer to
iASPP phosphorylated at one or more serine residues at the amino
terminus of the polypeptide. For example, in some embodiments, the
iASPP polypeptide comprises a phosphorylated serine residue
selected from the group consisting of Ser84 and Ser113 of human
iASPP (SEQ ID NO: 2). In some embodiments, Ser84 of human iASPP is
phosphorylated. In other embodiments, See 113 of human iASPP is
phosphorylated. In still other embodiments, both Ser84 and S113 of
human iASPP are phosphorylated. For the purposes of this aspect of
the invention, phosphorylation of iASPP at other residues such as a
tyrosine, and phosphorylation of residues outside of the
amino-terminus, are not considered in the measurement or evaluation
of the "phosphorylation state."
[0150] Some methods for measuring phosphorylated iASPP employ an
agent that binds a phosphorylated form of iASPP with great
specificity and binding affinity. Other methods employ an agent
that selectively binds iASPP that is unphosphorylated at Ser84
and/or Ser113. Although exemplified with antibodies, other suitable
binding partners exist and can be used to practice the
invention.
[0151] In some embodiments, an antibody that binds iASPP and
preferentially binds a phosphorylated form of iASPP is used to
measure the amount of phosphorylated iASPP in a sample. To
determine a measurement of phosphorylated iASPP, a biological
sample from a mammalian subject is contacted with an iASPP antibody
that preferentially binds phosphorylated iASPP under conditions and
for a time sufficient to allow immunocomplexes to form.
Immunocomplexes formed between the iASPP antibody and
phosphorylated iASPP in the sample are then detected. The amount of
phosphorylated iASPP in the biological sample is quantitated by
measuring the amount of the immunocomplex formed between the
antibody and the phosphorylated iASPP. For example, the capture
antibody can be quantitatively measured if it has a detectable
label, or a secondary antibody can be used to quantify the
immunocomplex.
[0152] In some embodiments, the biological sample comprises
cancerous tissue. Cancerous tissue from a solid tumor (e.g., a
carcinoma, sarcoma, glioma or lymphoma) can be obtained by using
conventional tumor biopsy instruments and procedures. Endoscopic
biopsy, excisional biopsy, incisional biopsy, fine needle biopsy,
punch biopsy, shave biopsy and skin biopsy are examples of
recognized medical procedures that can be used by those of skill in
the art to obtain tumor samples for use in practicing the methods
described herein. Samples of cancerous lesions may be obtained by
resection, bronchoscopy, fine needle aspiration, bronchial
brushings, or from sputum, pleural fluid or blood. In some
variations, a tumor or tumor biopsy is obtained from a subject and
fluid from the tumor is removed, e.g., by centrifugation, and the
tumor fluid is used as the sample for measuring phosphorylated
iASPP.
[0153] In one aspect, measuring the phosphorylation state of iASPP
in a cancer cell comprises an analytical technique selected from
the group consisting of (a) an analytical technique that provides
an absolute measure of phosphorylated iASPP, (b) an analytical
technique that provides a relative measure of phosphorylated iASPP
to total iASPP in the cell and (c) an analytical technique that
provides a relative measure of phosphorylated iASPP to
unphosphorylated iASPP in the cell.
[0154] An exemplary method for determining the absolute measure of
phosphorylated iASPP in the cancer cell comprises contacting the
cancer cell, or iASPP isolated from the cancer cell, with binding
partner that specifically binds the phosphorylated iASPP, and
measuring the amount of phosphorylated iASPP from the amount of
complex formed between the iASPP and binding partner. Optionally,
the second binding partner comprises a detectable label and
measuring the amount of phosphorylated iASPP is achieved by
measuring the amount of the binding partner-iASPP complex
formed.
[0155] In some embodiments, the method comprises measuring total
iASPP in the cell and measuring unphorylated iASPP in the cell,
wherein a difference between total iASPP and unphosphorylated iASPP
provides an indication of the amount of phosphorylated iASPP in the
cell.
[0156] Conditions for incubating an antibody with a test sample
vary. Incubation conditions depend on the format employed in the
assay, the detection methods employed, and the type and nature of
the antibody used in the assay. One skilled in the art will
recognize that any one of the commonly available immunological
assay formats can readily be adapted to employ the antibodies of
the present invention. Examples of such assays can be found in
Chard, T., An Introduction to Radioimmunoassay and Related
Techniques, Elsevier Science Publishers, Amsterdam, The Netherlands
(1986); Bullock, G. R. et al., Techniques in Immunocytochemistry,
Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2 (1983), Vol. 3
(1985); Tijssen, P., Practice and Theory of immunoassays:
Laboratory Techniques in Biochemistry and Molecular Biology,
Elsevier Science Publishers, Amsterdam, The Netherlands (1985). The
tests of the present invention include cells, protein extracts of
cells, or biological fluids such as, blood, serum, and plasma. The
test sample used in the above-described method will vary based on
the assay format, nature of the detection method and the tissues,
cells or extracts used as the sample to be assayed. Methods for
preparing protein extracts or membrane extracts of cells are well
known in the art and can be readily be adapted in order to obtain a
sample which is compatible with the system utilized.
[0157] In addition, such assays may be useful in evaluating the
efficacy of a particular therapeutic treatment regime in animal
studies, in clinical trials, or in monitoring the treatment of an
individual patient.
[0158] In some embodiments, the iASPP polypeptide or the binding
partner is attached to a solid support, and binding is detected by
detecting a complex between the polypeptide and the binding partner
on the solid support. The binding partner optionally comprises a
detectable label and binding is detected by detecting the label in
the polypeptide-binding partner complex.
[0159] A relative measure of phosphorylated iASPP to total iASPP in
the cancer cell can be determined by contacting the cancer cell, or
iASPP isolated from the cancer cell, with a first binding partner
and a second binding partner, wherein the first binding partner
binds iASPP and the second binding partner specifically binds the
phosphorylated iASPP. A relative measure of phosphorylation is
obtained by comparing the amount of complex formed between iASPP
and the first and second binding partners. Optionally, the first
binding partner comprises a first detectable label and the second
binding partner comprises a second detectable label, to facilitate
measuring the phosphorylation state of iASPP by measuring the
amount of the second binding partner-iASPP complex formed and
measuring total iASPP by measuring the amount of first binding
partner-iASPP complex formed. In some embodiments, the
phosphorylated iASPP recognized by the second binding partner
comprises one or more phosphorylated serine residues selected from
the group consisting of Ser84 and Ser113.
[0160] The method comprises, in some embodiments, contacting the
cancer cell, or protein isolated from the cancer cell, with a
binding partner that preferentially binds iASPP in which one or
more serine residues selected from serine 84 (Ser84, SEQ ID NO: 2)
and serine 113 (Ser113, SEQ ID NO: 2) is not phosphorylated (to
permit evaluation of the relative amount of total iASPP that is
unphosphorylated, as an indirect measure of the amount that is
phosphorylated).
[0161] The measuring steps described herein optionally comprise
isolating iASPP from the cancer cell, and measuring one or more
phosphorylated serine residues in the isolated iASPP. In some
variations, the iASPP is cleaved, and phosphorylation is measured
in one or more of the cleaved peptides. The cleaved peptides may be
separated by electrophoresis based upon size (e.g., by SDS-PAGE or
sizing gel). Other separation techniques may include aqueous
two-phase partitioning and non-denaturing agarose gel
electrophoresis separation. In other embodiments, separation
employs denaturing system such as an isoelectric focusing (IEF)
gel, capillary electrophoresis, or isotachyphoresis. Alternatively
or additionally, two-dimensional electrophoretic analysis may be
used (e.g., Wilkins et al., Proteome Research: New Frontiers in
Functional Genomics, Springer-Verlag, Berlin, 1997). Proteins can
be visualized on such gels using any of various stains known in the
art (e.g., Trypan Blue or SyproRuby dye). In some embodiments, the
method comprises measuring an absolute or relative amount of each
iASPP band as a measure of iASPP phosphorylation state. Mass
spectrometry technique can be used to quantify phosphorylated and
unphosphorylated peptides to provide a measure of iASPP
phosphorylation.
[0162] A relative measure of phosphorylated iASPP to
unphosphorylated iASPP in the cancer cell can be determined by, for
example, separating phorphorylated and unphosphorylated iASPP in
the cancer cell, and measuring the amounts of separated
phosphorylated and unphosphorylated iASPP relative to each other.
For example, in some embodiments, the separating step comprises
contacting the cancer cell, or iASPP isolated from the cancer cell,
with a binding partner that binds unphosphorylated iASPP and the
second binding partner specifically binds the phosphorylated iASPP,
and isolating formed phosphorylated iASPP-binding partner
complex.
[0163] The separation step can be accomplished in a variety of
ways. For example, at least one of the components can be
immobilized on a solid substrate, from which the unbound components
may be easily separated. The solid substrate can be made of a wide
variety of materials and in a wide variety of shapes, such as a
microtiter plate, a microbead, a dipstick, or a resin particle.
Ideally, the substrate provides maximum signal to noise ratios, to
minimize background binding.
[0164] In one example, separation is achieved by removing a bead or
dipstick from a reservoir, emptying or diluting a reservoir such as
a microtiter plate well, rinsing a bead, particle, chromatographic
column or filter with a wash solution or solvent. The separation
step can include multiple rinses or washes. For example, when the
solid substrate is a microtiter plate, the wells can be washed
several times with a washing solution, which typically includes
those components of the incubation mixture that do not participate
in specific bindings such as salts, buffer, detergent, non-specific
protein. Where the solid substrate is a magnetic bead, the beads
can be washed one or more times with a washing solution and
isolated using a magnet.
[0165] Detection of the presence or absence of a polypeptide
comprising an iASPP-binding partner complex can be achieved using
any method known in the art. For example, the transcript resulting
from a reporter gene transcription assay of an iASPP polypeptide
interacting with a target molecule (e.g., binding partner)
typically encodes a directly or indirectly detectable product (such
as .beta.-galactosidase activity, luciferase activity, and the
like). For cell free binding assays, one of the components usually
includes, or is coupled to, a detectable label. A wide variety of
labels can be used, such as those that provide direct detection
(such as radioactivity, luminescence, optical or electron density)
or indirect detection (such as epitope tag such as the FLAG
epitope, enzyme tag such as horseradish peroxidase). The label can
be bound to an iASPP binding partner, or incorporated into the
structure of the binding partner.
[0166] A variety of methods can be used to detect the label,
depending on the nature of the label and other assay components.
For example, the label can be detected while bound to the solid
substrate or subsequent to separation from the solid substrate.
Labels can be directly detected through optical or electron
density, radioactive emissions, nonradiative energy transfers or
indirectly detected with antibody conjugates, or strepavidin-biotin
conjugates. Methods for detecting the labels are well known in the
art.
[0167] As described herein, the phosphorylation state of iASPP in
the cancer cell is useful to diagnose metastatic potential of a
cancer. In some embodiments, the diagnosing comprises comparing the
amount of phosphorylated iASPP in the cancer cell with a
measurement of phosphorylated iASPP from healthy cells of the same
type as the cancer cell, wherein increased iASPP phosphorylation in
the cancer cell relative to the healthy cell indicates that the
cancer is at increased risk for metastases. In other embodiments,
the diagnosing comprises comparing the percentage of iASPP that is
phosphorylated in the cancer cell with a calculation of the
percentage of iASPP that is phosphorylated in a healthy cell of the
same type as the cancer cell, wherein increased percentage of
phosphorylated iASPP in the cancer cell indicates that the cancer
is at increased risk for metastases.
[0168] In any of the methods described herein, the binding partner
preferentially binds iASPP in which one or more serine residues
selected from serine 84 (Ser84 of SEQ ID NO: 2) and serine 113
(Ser113 of SEQ ID NO: 2) is phosphorylated, and measuring the
phosphorylation state by measuring the amount of protein-binding
partner complex formed to provide a measure of iASPP that is
phosphorylated at one or more of the serine residues.
[0169] In some variations, the complete iASPP polypeptide is
employed in the assay. However, evidence, including that described
below, indicates that cyclinB1/cdk1 phosphorylates serine at an
amino-terminal portion of iASPP, and that it is unnecessary to use
the complete iASPP for assays to measure phosphorylation. The term
"iASPP amino-terminal domain" ("N-iASPP") refers to a portion of
iASPP that is sufficient to exhibit the activity (e.g., interaction
with the kinase to receive phosphorylation of a serine). It is
possible to determine a minimum effective iASPP amino-terminal
domain by screening deletion fragments of iASPP according to kinase
assay procedures described herein. The iASPP amino-terminal domain
can be fused to other sequences to make fusion proteins, and/or
contain additional moieties such as labels and tags. Similarly, it
is possible to use an iASPP substrate to screen for fragments of
the kinase that is effective to phosphorylate iASPP for practicing
methods of the invention.
[0170] The phosphorylation state of iASPP can be determined in a
full length iASPP amino acid sequence or in fragments of the iASPP
amino acid sequence that include Ser84 and/or SER113. In one
aspect, the iASPP polypeptide comprises an amino-terminal domain
fragment of SEQ ID NO: 2, wherein the amino-terminal amino acid of
the fragment is selected from the group consisting of residues 1-83
of SEQ ID NO: 2, and wherein the carboxy terminal amino acid of the
fragment is selected from the group consisting of residues 113-500
of SEQ ID NO: 2. In some embodiments, the iASPP polypeptide
comprises amino acids 1-240 of SEQ ID NO: 2 or amino acids 1-828 of
SEQ ID NO: 2. In other aspects, the polypeptide comprising an iASPP
amino-terminal domain comprises amino acids 81-130 of SEQ ID NO: 2.
In another aspect, the polypeptide comprising an iASPP
amino-terminal domain comprises amino acids 1-478 of SEQ ID NO:
2.
[0171] In various embodiments, the iASPP amino terminal domain
optionally includes at least one phosphorylated serine between
residues 84 and 113 (inclusive) of SEQ ID NO: 2. For example, in
some embodiments, the iASPP amino terminal domain comprises amino
acids 81-130 of SEQ ID NO: 2, wherein the serines at positions 84
and 113 of SEQ ID NO: 2 are phosphorylated. In other embodiments,
the iASPP amino terminal domain comprises amino acids 81-130 of SEQ
ID NO: 2, wherein the serine at position 84 of SEQ ID NO: 2 is
phosphorylated. In still other embodiments, the iASPP amino
terminal domain comprises amino acids 81-130 of SEQ ID NO: 2,
wherein the serine at position 113 of SEQ ID NO: 2 is
phosphorylated.
[0172] Phosphorylated serine levels can be measured in situ or in
cells isolated from biological samples by standard in vitro
techniques well known in the art, such as enzyme-linked
immunosorbant assay (ELISA), radio immunoassay (RIA), Western blot,
or immunofluoroescence-based assays. iASPP activity can also be
measured in biological samples using fluorescent microscopy with
fluorescently labeled anti-iASPP and anti-phosphoserine antibodies.
The detection is correlated, for example, by a brighter staining
signal in a fluorescent microscopy assay, the presence of more
staining in a fluorescent microscopy assay, or by decreased fluid
levels of phosphorylated iASPP as detected by Western blot, ELISA,
RIA or other immunofluoroescence-based assays, such as fluorescence
resonance electron transfer (FRET). As described herein, it is
possible to measure phosphorylated iASPP using antibodies that
specifically recognize iASPP and differentiate between a
phosphorylated versus unphosphorylated form. Alternatively, it may
be possible to measure phosphorylation of iASPP by
isolating/measuring total iASPP (e.g., with an antibody that
recognizes an epitope unaffected by phosphorylation and using a
phosphoserine antibody or other tool that permits quantification of
phosphorylation.
[0173] High-content screens (HCS) provide for analysis of multiple
parameters in a single screening assay. For example,
phosphorylation of a polypeptide comprising an iASPP amino-terminal
domain may be measured using phosphoserine-specific antibodies
fluorescing in a particular excitation channel (e.g., Alexa Fluor
488 excites at 488 nm).
[0174] An anti-phosphoserine antibody or anti-phosphorylated iASPP
antibody suitable for use in the method of the invention may
comprise a label, such as a radioisotope, a fluorophore, a
fluorescing protein (e.g., natural or synthetic green fluorescent
proteins), a dye, an enzyme, a substrate, or the like. The label is
a compound or moiety known in the art to be useful as a label,
including biotin molecules, alkaline phosphatase, fluorophores
(e.g., fluoroisothiocyanate, phycoerythrin, Texas red, Alexa Fluor
stains, and other fluorescent dyes well known in the art),
radioisotopes (e.g., 3H, Europium3+, 32P), genetically engineered
peptide tags such as a histidine (His6) tag linked to the
aggregating polypeptide, a myc-tag, a Hemagluttinin tag, and the
like. Biotin, fluorophores, and other contemplated small molecules
comprising a label can be linked to the polypeptide of the
invention by means well-known in the art such as a commercially
produced Biotinylation kit (Sigma Chem. Co., St. Louis, Mo.), or
alternative methods commonly used in organic chemistry to attach a
small molecule to a peptide or protein (see e.g., Current Protocols
in Protein Chemistry, John Wiley & Sons, 2001). Genetically
engineered tags, e.g., His6 and myc-tags, are operably linked to
the polypeptide of the invention using standard recombinant DNA
methods well known in the art (see e.g., Current Protocols in
Molecular Biology, supra), or using conventional peptide synthesis
techniques. Such labels facilitate quantitative detection with
standard laboratory machinery and techniques.
[0175] B. Intracellular localization of iASPP
[0176] As described herein, intracellular localization of iASPP in
a cancer cell is indicative of whether the cancer cell is at an
increased risk for metastasis. The term "intracellular
localization" as used herein refers to the location of iASPP within
the cell. In some cells, iASPP is located within the cytoplasm of
the cell. In other cells, the iASPP is located within the nucleus
of the cell or is divided between the cytoplasm and nucleus. As
shown herein, increased nuclear localization of iASPP is associated
with increased risk of metastasis. Without intended to be limited
to any particular theory, the nuclear iASPP is believed to inhibit
tumor suppressive activity of p53. Intracellular localization can
be measured by methods known in the art, such as
immunohistochemical analysis.
[0177] In some embodiments, the amount of iASPP within the
cytoplasm is measured. In some embodiments, the amount of iASPP
within the nucleus is measured.
[0178] In some embodiments, intracellular localization is measured
by an analytical technique selected from the group consisting of an
analytical technique that provides an absolute measure of iASPP in
the nucleus of the cancer cell, analytical technique that provides
a relative measure of iASPP in the nucleus to total iASPP in the
cell, and an analytical technique that provides a relative measure
of iASPP in the nucleus to iASPP in the cytoplasm in the cell. In
some embodiments, the measuring comprises immunohistochemical
staining of the cancer cell with an antibody that specifically
binds iASPP.
[0179] Intracellular localization is measured, in some embodiments,
by comparing the measure of iASPP in the nucleus of the cancer cell
with a measure of iASPP in a healthy cell of the same type as the
cancer cell, wherein increased iASPP in the nucleus of the cancer
cell compared to the healthy cell indicates an increased metastatic
potential for the cancer.
[0180] Methods of measuring intracellular localization as described
herein optionally comprise contacting the cancer cell with a
nuclear-specific marker. Nuclear-specific markers are known in the
art and include, but are not limited to, Hoechst 33258, Hoechst
33342, 4',6-Diamidino-2-phenylindole (DAPI);
4',6-diacarboxyamide-2-phenylindole (DCI); 7-Aminoactinomycin D
(7-AAD); Ethidium bromide; Ethidium homodimer; Propidium iodide
(PI); Antraquinone dye (DRAQ5); TO-PRO-1; TO-PRO-3; TOTO-1; TOTO-3;
YO-PRO-1; YOYO-1; SYTOX Green; Acridine orange (AO) and Pyronin Y.
See, for example, Haughland, R. (1998) Handbook of fluorescent
probes and research chemicals, 6 Ed., Molecular Probes, Inc., the
disclosure of which is incorporated by reference in its
entirety.
[0181] In some embodiments, intracellular localization of iASPP is
measured in a section of a tumor tissue sample using procedures
described in, for example, Van Impe et al., (2008). A new role for
nuclear transport factor 2 and Ran: nuclear import of CapG. Traffic
(Copenhagen, Denmark) 9, 695-707, the disclosure of which is
incorporated herein by reference in its entirety. For example, in
some embodiments, the method comprises contacting a tissue sample
with an antibody that binds iASPP and labeled with a nuclear
specific marker and counting the number of cells in which
co-localization of the iASPP antibody with the nuclear specific
marker is detected. In another embodiment, the method comprises
containing a tissue sample with an antibody that specifically binds
iASPP, wherein the antibody comprises a detectable marker, and
contacting the tissue sample with a nuclear specific marker,
identifying iASPP within the nucleus of cells in the tissue sample
by detecting the presence of an iASPP-antibody complex within the
nucleus of the cells and counting the number of cells in which the
iASPP-antibody complex is identified in the nucleus of the
cells.
[0182] The methods described herein optionally comprise measuring
the mitotic index of the cancer using methods known in the art,
such as those described in Example 3.
[0183] II. Binding Partners
[0184] Binding partners (e.g., antibodies) that bind to
phosphorylated forms of iASPP are also contemplated. In one aspect,
described herein is an iASPP binding partner selected from the
group consisting of (a) an antibody that specifically binds iASPP
polypeptide and preferentially binds to iASPP having a
phosphorylated serine compared to binding to iASPP without the
phosphorylated serine, wherein said serine residue corresponds to a
serine selected from the group consisting of Ser84 and Ser113 of
the human iASPP amino acid sequence of SEQ ID NO: 2; (b) a fragment
of (a) that specifically binds iASPP polypeptide and preferentially
binds to iASPP having the phosphorylated serine; and (c) a
polypeptide that comprises (b) and that specifically binds iASPP
polypeptide and preferentially binds to iASPP having the
phosphorylated serine.
[0185] In some embodiments, the antibody binds to an epitope of the
phosphorylated peptide comprising a portion of the amino acid
sequence SRGpSPRKAATDGADTPFGRDESAPTLHPYSPLpSPKGRPSSPRTPLYLQPD
(amino acids 81-130 of SEQ ID NO: 2), where pS is a phosphorylated
serine. In some embodiments, the antibody binds to an epitope of
the phosphorylated peptide that comprises at least 4 (or at least
5, at least 6, at least 7, at least 8, at least 9, at least 10, at
least 11, at least 12, at least 13, at least 14, at least 15, at
least 16, at least 17, at least 18, at least 19, at least 20, at
least 25, at least 30 or more) of amino acids 81-130 of SEQ ID NO:
2. For example, in some variations, the antibody binds to an
epitope of the phosphorylated iASPP peptide that comprises amino
acids 81-86 of SEQ ID NO: 2, or amino acids 81-90 of SEQ ID NO: 2,
or amino acids 81-95 of SEQ ID NO: 2, or amino acids 81-100 of SEQ
ID NO: 2, or amino acids 83-89 of SEQ ID NO: 2, or amino acids
83-94 of SEQ ID NO: 2, or amino acids 83-100 of SEQ ID NO: 2, or
amino acids or amino acids 110-115 of SEQ ID NO: 2, or all or some
of amino acids 107-118 of SEQ ID NO: 2, or amino acids 110-120 of
SEQ ID NO: 2, or amino acids 110-125 of SEQ ID NO: 2 or amino acids
111-122 of SEQ ID NO: 2. In one variation, the antibody binds to a
phosphorylated iASPP peptide that comprises a conformational
epitope. In such variations, for example, the antibody binds to
multiple epitopes within amino acids 81-130 of SEQ ID NO: 2.
[0186] In another aspect, described herein is an iASPP binding
partner selected from the group consisting of (a) an antibody that
specifically binds iASPP polypeptide and preferentially binds iASPP
in which one or more serine residues selected from serine 84
(Ser84, SEQ ID NO: 2) and serine 113 (Ser113, SEQ ID NO: 2) is not
phosphorylated. compared to binding to iASPP having the
phosphorylated serine, (b) a fragment of (a) that specifically
binds iASPP polypeptide and preferentially binds to iASPP having
the phosphorylated serine; and (c) a polypeptide that comprises (b)
and that specifically binds iASPP polypeptide and preferentially
binds to iASPP without the phosphorylated serine. In some
embodiments, the antibody is LX128.5 (i.e., an antibody that binds
to an epitope comprising all or some of amino acids 111-122 of SEQ
ID NO: 2, including serine 113).
[0187] In some embodiments, the antibody is selected from the group
of antibodies consisting of LX49.3, LX128.1, LX128.2, LX128.3,
LX128.4, LX128.5, LX128.6, LX128.7, LX128.9 and LC128.10. In one
embodiments, the antibody is LX128.5.
[0188] An antibody or antibody fragment that specifically binds to
a phosphorylated form of iASPP can be identified, for example, by
immunoassays, BIAcore, or other techniques known to those of skill
in the art. An antibody or antibody fragment binds specifically to
phosphorylated iASPP when it binds to the phosphorylated iASPP with
higher affinity than to any cross-reactive protein (especially
unphosphorylated iASPP) as determined using experimental
techniques, such as RIAs and ELISAs. Typically, a specific or
selective reaction will be at least twice the background signal or
noise and more typically more than 10 times background. In one
embodiment, the antibody specifically binds to an epitope that
includes one or more phosphorylated serine residues (Ser84 or
Ser113) of iASPP. In another certain embodiment, the antibody
specifically binds to an epitope that includes only one
phosphorylated serine residue of iASPP.
[0189] Antibodies useful for detecting peptides comprising
phosphorylated serine residues are generated using techniques well
known in the art. In some variations, phosphorylated iASPP or
phosphorylated iASPP fragments are used as an antigen to immunize
an animal, or to select antibodies from a library. The antibodies
so selected are then screened against unphosphorylated iASPP, to
identify antibodies that selectively bind the phosphorylated, but
not the unphosphorylated, form. An analogous process, starting with
unphosphorylated iASPP, is used to select antibodies specific for
the unphosphorylated form.
[0190] The invention contemplates use of antibodies (e.g.,
monoclonal and polyclonal antibodies, single chain antibodies,
chimeric antibodies, bifunctional/bispecific antibodies, humanized
antibodies, human antibodies, and complementarity determining
region (CDR)-grafted antibodies, including compounds that include
CDR sequences specifically recognizing a polypeptide of the
invention and specific for polypeptides of interest to the
invention, especially phosphorylated serine on the iASPP
polypeptide). Antibodies can be human antibodies which are produced
and identified according to methods described in WO 93/11236, which
is incorporated herein by reference in its entirety. Antibody
fragments, including Fab, Fab', F(ab').sub.2, and Fv, and
single-chain antibodies are also provided by the invention. The
term "specific for," when used to describe antibodies of the
invention, indicates that the variable regions of the antibodies of
the invention recognize and bind the polypeptide of interest with a
detectable preference (i.e., able to distinguish the polypeptide of
interest from other known polypeptides of the same family, by
virtue of measurable differences in binding affinity, despite the
possible existence of localized sequence identity, homology, or
similarity between family members). It will be understood that
specific antibodies may also interact with other proteins (for
example, S. aureus protein A or other antibodies in ELISA
techniques) through interactions with sequences outside the
variable region of the antibodies, and in particular, in the
constant region of the molecule. Screening assays to determine
binding specificity of an antibody of the invention are well known
and routinely practiced in the art. For a comprehensive discussion
of such assays, see Harlow et al. (Eds), Antibodies A Laboratory
Manual; Cold Spring Harbor Laboratory; Cold Spring Harbor, NY
(1988), Chapter 6. Antibodies of the invention can be produced
using any method well known in the art.
[0191] Various procedures known in the art may be used for the
production of polyclonal antibodies to peptides comprising
phosphorylated serine. For the production of antibodies, various
host animals (including but not limited to rabbits, mice, rats,
hamsters, and the like) are immunized by injection with a
phosphorylated iASPP protein or peptide. Various adjuvants may be
used to increase the immunological response, depending on the host
species, including but not limited to Freund's (complete and
incomplete) adjuvant, mineral gels such as aluminium hydroxide,
surface active substances such as lysolecithin, pluronic polyols,
polyanions, oil emulsions, keyhole limpet hemocyanins,
dinitrophenol, and potentially useful human adjuvants such as BCG
(Bacillus Calmette-Guerin) and Corynebacterium parvum.
[0192] A monoclonal antibody to a phosphorylated epitope of iASPP
may be prepared by using any technique which provides for the
production of antibody molecules by continuous cell lines in
culture. These include but are not limited to the hybridoma
technique originally described by Kohler et al., Nature, 256:
495-497 (1975), and the more recent human B-cell hybridoma
technique [Kosbor et al., Immunology Today, 4: 72 (1983)] and the
EBV-hybridoma technique [Cole et al., Monoclonal Antibodies and
Cancer Therapy, Alan R Liss, Inc., pp. 77-96 (1985), all
specifically incorporated herein by reference]. Antibodies against
phosphorylated iASPP also may be produced in bacteria from cloned
immunoglobulin cDNAs. With the use of the recombinant phage
antibody system it may be possible to quickly produce and select
antibodies in bacterial cultures and to genetically manipulate
their structure.
[0193] When the hybridoma technique is employed, myeloma cell lines
may be used. Such cell lines suited for use in hybridoma-producing
fusion procedures preferably are non-antibody-producing, have high
fusion efficiency, and exhibit enzyme deficiencies that render them
incapable of growing in certain selective media which support the
growth of only the desired fused cells (hybridomas). For example,
where the immunized animal is a mouse, one may use P3-X63/Ag8,
P3-X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14, FO, NSO/U, MPC-11,
MPC11-X45-GTG 1.7 and S194/5XX0 Bul; for rats, one may use
R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2,
LICR-LON-HMy2 and UC729-6 are all useful in connection with cell
fusions. It should be noted that the hybridomas and cell lines
produced by such techniques for producing the monoclonal antibodies
are contemplated compositions of the present invention.
[0194] In addition to the production of monoclonal antibodies,
techniques developed for the production of "chimeric antibodies,"
the splicing of mouse antibody genes to human antibody genes to
obtain a molecule with appropriate antigen specificity and
biological activity can be used [Morrison et al., Proc. Natl. Acad.
Sci. 81:6851-6855 (1984); Neuberger et al., Nature 312:604-608
(1984); Takeda et al., Nature 314:452-454 (1985)]. Alternatively,
techniques described for the production of single-chain antibodies
(U.S. Pat. No. 4,946,778) can be adapted to produce
phosphorylated-iASPP peptide-specific single chain antibodies.
[0195] Antibody fragments which contain the idiotype of the
molecule may be generated by known techniques. For example, such
fragments include, but are not limited to, the F(ab').sub.2
fragment which may be produced by pepsin digestion of the antibody
molecule; the Fab' fragments which may be generated by reducing the
disulfide bridges of the F(ab').sub.2 fragment, and the two Fab
fragments which may be generated by treating the antibody molecule
with papain and a reducing agent.
[0196] Rapid, large scale recombinant methods for generating
antibodies may be employed, such as phage display [Hoogenboom et
al., J. Mol. Biol. 227: 381, (1991); Marks et al., J. Mol. Biol.
222: 581, (1991)] or ribosome display methods, optionally followed
by affinity maturation [see, e.g., Ouwehand et al., Vox Sang
74(Suppl 2):223 232 (1998); Rader et al., Proc. Natl. Acad. Sci.
USA 95:8910 8915 (1998); Dall'Acqua et al., Curr. Opin. Struct.
Biol. 8:443 450, (1998)]. Phage-display processes mimic immune
selection through the display of antibody repertoires on the
surface of filamentous bacteriophage, and subsequent selection of
phage by their binding to an antigen of choice. One such technique
is described in WO 99/10494, which describes the isolation of high
affinity and functional agonistic antibodies for MPL and msk
receptors using such an approach.
[0197] "Antibody fragments" comprise a portion of an intact
immunoglobulin, e.g., an antigen binding or variable region of the
intact antibody, and include multispecific (bispecific,
trispecific, etc.) antibodies formed from antibody fragments.
Fragments of immunoglobulins may be produced by recombinant DNA
techniques or by enzymatic or chemical cleavage of intact
antibodies.
[0198] Nonlimiting examples of antibody fragments include Fab,
Fab', F(ab').sub.2, Fv (variable region), domain antibodies (dAb,
containing a V.sub.H domain) (Ward et al., Nature 341:544-546,
1989), complementarity determining region (CDR) fragments,
single-chain antibodies (scFv, containing V.sub.H and V.sub.L
domains on a single polypeptide chain) (Bird et al., Science
242:423-426, 1988, and Huston et al., Proc. Natl. Acad. Sci. USA
85:5879-5883, 1988, optionally including a polypeptide linker; and
optionally multispecific, Gruber et al., J. Immunol. 152: 5368
(1994)), single chain antibody fragments, diabodies (V.sub.H and
V.sub.L domains on a single polypeptide chain that pair with
complementary V.sub.L and V.sub.H domains of another chain) (EP
404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci.
USA, 90:6444-6448 (1993)), triabodies, tetrabodies, minibodies
(scFv fused to CH3 via a peptide linker (hingeless) or via an IgG
hinge) (Olafsen, et al., Protein Eng Des Sel. 2004 April;
17(4):315-23), maxibodies (bivalent scFvs covalently attached to
the Fc region of an immunoglobulin; Fredericks et al, Protein
Engineering, Design & Selection, 17:95-106 (2004) and Powers et
al., Journal of Immunological Methods, 251:123-135 (2001)); linear
antibodies (tandem Fd segments (V.sub.H-C.sub.H1-V.sub.H-C.sub.H1)
(Zapata et al., Protein Eng., 8(10):1057-1062 (1995)); chelating
recombinant antibodies (crAb, which can bind to two adjacent
epitopes on the sane antigen) (Neri et al., J Mol Biol. 246:367-73,
1995), bibodies (bispecific Fab-scFv) or tribodies (trispecific
Fab-(scFv)(2)) (Schoonjans et al., J Immunol. 165:7050-57, 2000;
Willems et al., J Chromatogr B Analyt Technol Biomed Life Sci.
786:161-76, 2003), intrabodies (Biocca, et al., EMBO J. 9:101-108,
1990; Colby et al., Proc Natl Acad Sci USA. 101:17616-21, 2004)
which may also comprise cell signal sequences which retain or
direct the antibody intracellularly (Mhashilkar et al, EMBO J
14:1542-51, 1995; Wheeler et al., FASEB J. 17:1733-5, 2003),
transbodies (cell-permeable antibodies containing a protein
transduction domain (PTD) fused to scFv (Heng et al., Med
Hypotheses. 64:1105-8, 2005), nanobodies (approximately 15 kDa
variable domain of the heavy chain) (Cortez-Retamozo et al., Cancer
Research 64:2853-57, 2004), small modular immunopharmaceuticals
(SMIPs) (WO03/041600, U.S. Patent publication 20030133939 and US
Patent Publication 20030118592), an antigen-binding-domain
immunoglobulin fusion protein, a camelized antibody (in which VH
recombines with a constant region that contains hinge, C.sub.H1,
C.sub.H2 and C.sub.H3 domains) (Desmyter et al., J. Biol. Chem.
276:26285-90, 2001; Ewert et al., Biochemistry 41:3628-36, 2002;
U.S. Patent Publication Nos. 20050136049 and 20050037421), a
V.sub.HH containing antibody, heavy chain antibodies (HCAbs,
homodimers of two heavy chains having the structure
H.sub.2L.sub.2), or variants or derivatives thereof, and
polypeptides that contain at least a portion of an immunoglobulin
that is sufficient to confer specific antigen binding to the
polypeptide, such as a CDR sequence, as long as the antibody
retains the desired biological activity. Such antigen fragments may
be produced by the modification of whole antibodies or synthesized
de novo using recombinant DNA technologies or peptide
synthesis.
[0199] III. Inhibitor Peptides
[0200] Another aspect of the invention includes peptides that
inhibit iASPP phosphorylation. When such a peptide is introduced
into a system that contains iASPP and a kinase capable of
phosphorylating iASPP, the peptide inhibits the kinase from
phosphorylating iASPP. Such inhibition can be measured by, e.g., a
slowing of the rate at which the iASPP is phosphorylated; or by a
reduced amount of phosphorylated iASPP in an equilibrium state. In
some embodiments, the kinase is a serine kinase such as
cyclinB1/cdk1.
[0201] In some variations, the peptide has a structure that mimics
that portion of iASPP that is a kinase substrate, but does not
function in downstream signaling pathways of phosphorylated iASPP.
For example, the peptide may have a structure that binds
cyclinB1/cdk1 and is itself phosphorylated, but lacks structure
such as the C-terminal iASPP domain involved in downstream
signaling. In some variations, the peptide irreversibly or
reversibly binds the kinase to prevent the kinase from binding and
phosphorylating endogenous iASPP. In some embodiments, the peptide
has a structure that permits kinase binding, but lacks a structure
to receive phosphorylation, e.g., serine deletion variants of
iASPP/iASPP fragments. It will be appreciated that these variations
are not mutually exclusive from each other.
[0202] In one aspect, described herein is an isolated peptide
comprising an amino acid sequence selected from the group
consisting of (a) a fragment of the iASPP amino acid sequence set
forth in SEQ ID NO: 2, wherein the fragment binds to a kinase that
phosphorylates iASPP at a serine residue selected from the group
consisting of Ser84 and Ser113 of SEQ ID NO: 2; (b) an analog of
(a) wherein from 1-10 amino acids have been deleted, inserted, or
replaced with different amino acids, wherein said analog binds to
the kinase that phosphorylates iASPP at a serine residue selected
from the group consisting of Ser84 and Ser113 of SEQ ID NO: 2; (c)
a cyclized version of (a) or (b) that binds to the kinase that
phosphorylates iASPP at a serine residue selected from the group
consisting of Ser84 and Ser113 of SEQ ID NO: 2; wherein the peptide
binds to a kinase that phosphorylates iASPP at a serine residue
selected from the group consisting of Ser84 and Ser113 of SEQ ID
NO: 2, and wherein the peptide inhibits the kinase from
phosphorylating iASPP.
[0203] In one variation, the peptide comprises an amino acid
sequence at least 85% (or at least 90%, at least 91%, at least 92%,
at least 93%, at least 94%, at least 95%, at least 96%, at least
97%, at least 99%) or more identical to a fragment of SEQ ID NO: 2,
wherein the amino-terminal amino acid of the fragment is selected
from the group consisting of residues 1-83 of SEQ ID NO: 2, and
wherein the carboxy terminal amino acid of the fragment is selected
from the group consisting of residues 113-500 of SEQ ID NO: 2. In
other embodiments, the peptide comprises an amino acid sequence at
least 85% (or at least 90%, at least 91%, at least 92%, at least
93%, at least 94%, at least 95%, at least 96%, at least 97%, at
least 99%) or more identical to amino acids 1-240 of SEQ ID NO: 2
or amino acids 81-130 of SEQ ID NO: 2 or amino acids 107-118 of SEQ
ID NO: 2.
[0204] In another variation, the iASPP inhibitor peptides described
herein comprises a fragment of SEQ ID NO: 2 comprising at least 10,
at least 15, at least 20, at least 21, at least 22, at least 23, at
least 24, at least 25, at least 26, at least 27, at least 28, at
least 29, at least 30, at least 31, at least 32, at least 33, at
least 34, at least 35, at least 36, at least 37, at least 38, at
least 39, at least 40, at least 41, at least 42, at least 43, at
least 44, at least 45, at least 46, at least 47, at least 48, at
least 49, at least 50, at least 51, at least 52, at least 53, at
least 54, at least 55, at least 56, at least 57, at least 58, at
least 59, at least 60, at least 61, at least 62, at least 63, at
least 64, at least 65, at least 66, at least 67, at least 68, at
least 69, at least 70, at least 71, at least 72, at least 73, at
least 74, at least 75, at least 76, at least 77, at least 78, at
least 79, at least 80, at least 81, at least 82, at least 83, at
least 84, at least 85, at least 86, at least 87, at least 88, at
least 89, at least 90, at least 91, at least 92, at least 93, at
least 94, at least 95, at least 96, at least 97, at least 98, at
least 99, at least 100 or more amino acids of SEQ ID NO: 2, wherein
said fragment binds to a kinase that phosphorylates wild type iASPP
at a serine residue selected from the group consisting of Ser84 and
Ser113 of SEQ ID NO: 2.
[0205] Another aspect of the invention includes peptides that
inhibit translocation of iASPP into the nucleus of a cancer cell,
or a cancer cell line in which iASPP has been shown to localize to
the nucleus. Without intending to be limited to any particular
theory, the peptide is believed to operate in this manner by
mimicking the self-interaction of an amino terminal region of iASPP
with a carboxy terminal region of iASPP. In one variation, the
peptide comprises an amino acid sequence at least 85% (or at least
90%, at least 91%, at least 92%, at least 93%, at least 94%, at
least 95%, at least 96%, at least 97%, at least 99%) or more
identical to amino acids 1-240 of SEQ ID NO: 2 with the proviso
that the serine at residues 84 and 113 of SEQ ID NO: 2 are not
phosphorylated, or a fragment thereof that mimics the
self-interaction of an amino terminal fragment of iASPP with a
carboxy terminal fragment of iASPP. In some embodiments, the
peptide does not include S84D or S113D mutations. Data presented
herein shows that iASPP with these mutations mimics phosphorylated
wildtype iASPP.
[0206] Variant iASPP inhibitor peptides are also contemplated.
Variant peptides may differ in amino acid sequence by one or more
substitutions, additions, deletions, truncations which may be
present in any combination. In one aspect, the modification is a
conservative substitution. Such substitutions are those that
substitute a given amino acid by another amino acid of like side
chain characteristics, such as being acidic, basic, polar,
nonpolar, aliphatic, aromatic, etc. The following non-limiting list
of amino acids are considered conservative replacements (similar):
a) alanine, serine, and threonine; b) glutamic acid and asparatic
acid; c) asparagine and glutamine d) arginine and lysine; e)
isoleucine, leucine, methionine and valine and f) phenylalanine,
tyrosine and tryptophan.
[0207] The present disclosure includes iASPP inhibitor peptides, as
well as synthetic examples of the proteins described herein. In
addition, analogs (non-peptide organic molecules), derivatives
(chemically functionalized peptide molecules obtained starting with
the disclosed peptide sequences) and variants (homologs) of these
proteins can be utilized in the methods described herein. For
example, iASPP inhibitor peptides that include modifications, but
retain the ability to interfere with the binding of wild type iASPP
to an iASPP binding partner or the phosphorylation of iASPP are
also contemplated. The peptides disclosed herein include a sequence
of amino acids, which can be either L- or D-amino acids,
naturally-occurring and otherwise.
[0208] Peptides can be modified by a variety of chemical techniques
to produce derivatives having essentially the same activity as the
unmodified peptides, and optionally having other desirable
properties. For example, carboxylic acid groups of the protein,
whether carboxyl-terminal or side chain, may be provided in the
form of a salt of a pharmaceutically-acceptable cation or
esterified to form a C.sub.1-C.sub.16 ester, or converted to an
amide of formula NR1R2 wherein R1 and R2 are each independently H
or C.sub.1-C.sub.16 alkyl, or combined to form a heterocyclic ring,
such as a 5- or 6-membered ring. Amino groups of the peptide,
whether amino-terminal or side chain, may be in the form of a
pharmaceutically-acceptable acid addition salt, such as the HCl,
HBr, acetic, benzoic, toluene sulfonic, maleic, tartaric and other
organic salts, or may be modified to C.sub.1-C.sub.16 alkyl or
dialkyl amino or further converted to an amide.
[0209] Hydroxyl groups of the peptide side chains may be converted
to C.sub.1-C.sub.16 alkoxy or to a C.sub.1-C.sub.16 ester using
well-recognized techniques. Phenyl and phenolic rings of the
peptide side chains may be substituted with one or more halogen
atoms, such as fluorine, chlorine, bromine or iodine, or with
C.sub.1-C.sub.16 alkyl, C.sub.1-C.sub.16 alkoxy, carboxylic acids
and esters thereof, or amides of such carboxylic acids. Methylene
groups of the peptide side chains can be extended to homologous
C.sub.2-C.sub.4 alkylenes. Thiols can be protected with any one of
a number of well-recognized protecting groups, such as acetamide
groups. Those skilled in the art will also recognize methods for
introducing cyclic structures into the peptides of this invention
to select and provide conformational constraints to the structure
that result in enhanced stability. For example, a carboxyl-terminal
or amino-terminal cysteine residue can be added to the peptide, so
that when oxidized the peptide will contain a disulfide bond,
generating a cyclic peptide. Other peptide cyclizing methods
include the formation of thioethers and carboxyl- and
amino-terminal amides and esters.
[0210] Peptidomimetic and organomimetic embodiments are also within
the scope of the present disclosure, whereby the three-dimensional
arrangement of the chemical constituents of such peptido- and
organomimetics mimic the three-dimensional arrangement of the
peptide backbone and component amino acid side chains, resulting in
such peptido- and organomimetics of the proteins of this disclosure
having measurable or enhanced ability to bind an antibody. For
computer modeling applications, a pharmacophore is an idealized,
three-dimensional definition of the structural requirements for
biological activity. Peptido- and organomimetics can be designed to
fit each pharmacophore with current computer modeling software
(using computer assisted drug design or CADD). See Walters,
"Computer-Assisted Modeling of Drugs", in Klegerman & Groves,
eds., 1993, Pharmaceutical Biotechnology, Interpharm Press: Buffalo
Grove, Ill., pp. 165 174 and Principles of Pharmacology Munson
(ed.) 1995, Ch. 102, for descriptions of techniques used in CADD.
Also included within the scope of the disclosure are mimetics
prepared using such techniques.
[0211] Nucleic acids encoding the inhibitor peptides are another
aspect of the invention.
[0212] Vectors also are useful for "gene therapy" treatment
regimens, wherein a polynucleotide that encodes a polypeptide of
the invention is introduced into a subject in need of treatment
involving the modulation (decreased level or inhibition) of iASPP
phosphorylation, in a form that causes cells in the subject to
express the polypeptide of the invention in vivo. Gene therapy
aspects that are described in WO 2007/006573, the disclosure of
which is incorporated herein by reference, is also applicable
herein.
[0213] IV. Therapeutic Methods
[0214] Also described herein are methods of treatment comprising
obtaining a tumor or tumor biopsy from a mammalian subject,
measuring the phosphorylation state of iASPP in the tumor sample
and prescribing for or administering to the subject with elevated
iASPP phosphorylation a composition comprising a molecule that
suppresses expression or signaling activity of cdk1, a molecule
that inhibits phosphorylation of iASPP, an inhibitor of B-RafV600E,
and/or an inhibitor of mdm2-induced degradation of p53. In some
variations, the invention is simply the treatment steps without
first determining the phosphorylation state of iASPP in a cancer
cell of a mammalian subject.
[0215] In some embodiments, described herein are methods of
treating a subject identified as having elevated iASPP
phosphorylation comprising administering to the subject at least
one therapeutic selected from the group consisting of a molecule
that suppresses expression or signaling activity of cdk1, a
molecule that inhibits phosphorylation of iASPP, an inhibitor of
B-RafV600E, and an inhibitor of mdm2-induced degradation of p53. In
some embodiments, the method comprises administering at least two
therapeutics selected from the group consisting of a molecule that
suppresses expression or signaling activity of cdk1, a molecule
that inhibits phosphorylation of iASPP, an inhibitor of B-RafV600E,
and an inhibitor of mdm2-induced degradation of p53.
[0216] In one aspect, a method of treating a mammalian subject with
cancer is provided, wherein the method comprises administering to
the subject an inhibitor of cyclinB1/cdk1-induced phosphorylation
of iASPP; an inhibitor of mdm2-induced degradation of p53; and an
inhibitor of B-RafV600E. In some variations, the compounds are in
admixture and administered together. In some variations, the
inhibitors are packaged together, and at least two of the
inhibitors are not in admixture. The inhibitors are preferably
administered in amounts effective to induce death of cancer cells
in the mammalian subject.
[0217] Another aspect is a therapeutic regimen for treating cancer
in a mammalian subject. For example, in some embodiments, the
therapeutic regimen comprises measuring phosphorylation state
and/or intracellular localization of iASPP in a cancer cell from a
mammalian subject, screening the cancer cell for at least one
parameter selected from the group consisting of a p53 mutation that
reduces a tumor suppressive function of p53, an elevated
measurement of mdm2 protein or mRNA, and a B-RafV600E mutation, and
prescribing and/or administering a therapeutic regimen that
includes an inhibitor of iASPP phosphorylation for a subject with a
cancer characterized by elevated iASPP phosphorylation and/or iASPP
nuclear localization.
[0218] The prescribing and/or administering optionally comprises a
therapeutic regiment that includes an inhibitor of iASPP
phosphorylation and a molecule that suppresses expression or
signaling activity of cdk1.
[0219] The prescribing and/or administering optionally comprises a
therapeutic regimen that includes an inhibitor of iASPP
phosphorylation and an inhibitor of mdm2-induced degradation of
p53, for a subject with a cancer characterized by elevated iASPP
phosphorylation and/or iASPP nuclear localization and the absence
of a p53 mutation that reduces the tumor suppressive function of
p53.
[0220] In some embodiments, the prescribing and/or administering
comprises a therapeutic regimen that includes an inhibitor of iASPP
phosphorylation and an inhibitor of B-RafV600E, for a subject with
a cancer characterized by elevated iASPP phosphorylation and/or
iASPP nuclear localization and the presence of the B-Raf-V600E
mutation.
[0221] P53 mutations that reduce tumor suppressive function of p53
in a plurality of cancer are well known in the art. For review, see
Suzuki et al., J. Biomed. Biotechnol., 2011:978312, 2001, the
disclosure of which is incorporated here by reference in its
entirety. More than 26,000 somatic mutation data of p53 appear in
the international agency for research on cancer (IARC) TP53
database version R14 (www-p53.iarc.fr/).
[0222] In some embodiments, molecules that inhibit iASPP
phosphorylation include molecules that suppress expression or
signaling activity of cdk1 (i.e., a cdk1 inhibitor). Exemplary cdk1
inhibitors include, but are not limited to an antibody that binds
cdk1 and inhibits ligand-mediated stimulation of cdk1; an antisense
or interfering nucleic acid (e.g., antisense oligonucleotide;
micro-RNA, short interfering RNA) that inhibits cdk1 expression; an
antibody that binds cdk1; an antisense or interfering nucleic acid
(e.g., antisense oligonucleotide; micro-RNA, short interfering RNA)
that inhibits expression of cdk1, a small molecule that inhibits
cdk1 expression or signaling; a small molecule that inhibits cdk1
expression or signaling; and a molecule that inhibits cyclinB1 from
binding to cdk1.
[0223] In some embodiments, the cdk1 inhibitor is an antibody
(e.g., an antibody that binds to cdk1 and inhibits formation of the
cyclinB1/cdk1 complex). Such antibodies include, but are not
limited to, polyclonal, monoclonal, chimeric, single chain, Fab
fragments and fragments produced by a Fab expression library,
bifunctional/bispecific antibodies, humanized antibodies, CDR
grafted antibodies, human antibodies and antibodies which include
portions of CDR sequences specific for cdk1.
[0224] Neutralizing antibodies, i.e., those which may inhibit
formation of the cyclinB1/cdk1 complex or inhibit binding of the
complex to iASPP, are especially preferred. In a preferred
embodiment, the antibody is a monoclonal antibody. In some
embodiments, the antibody is a human or a humanized antibody.
Techniques described elsewhere herein are useful for the
preparation of antibodies for both detection and therapeutic
purposes.
[0225] Various small molecule cdk1 inhibitors are currently
undergoing clinical trials. See Malumbres et al., Nat. Rev. Canc.
9:153-166, 2009, Table 2 in particular. In some embodiments, the
molecule that suppresses expression or signaling activity of cdk1
includes, but is not limited to AT-7519, P276-00, R547,
Flavopiridol, UCN-01, BMS-387032, K00616A and CDKi277.
[0226] In some embodiments, the cdk1 inhibitor is an inhibitor
provided below in Table 1.
TABLE-US-00001 TABLE 1 Drug Name (CD, GN, Molecular Therapeutic BN)
Chemical Name/Description Mechanism Group Mechanism of Action
Scytonemin 3,3'-Bis(4- CDK1/Cyclin B1 Oncolytic Antiinflammatory
hydroxyphenylmethylene)-(1,1'- Inhibitors Drugs; Drugs; Antimitotic
bicyclopent[b]indole)-2,2'- Ultraviolet Drugs; CDK1/Cyclin B1
(3H,3'H)-dione Light Inhibitors; Checkpoint Absorbers Kinase 1
(Chk1) Inhibitors; Inhibitors of Signal Transduction Pathways; Myt1
Kinase Inhibitors; Polo-like Kinase Inhibitors; Protein Kinase PKC
beta 2 Inhibitors Checkpoint Kinase 1 (Chk1) Inhibitors Myt1 Kinase
Inhibitors Polo-like Kinase Inhibitors Protein Kinase PKC beta 2
Inhibitors N-[2-[2-[4-(N,N- CDK1/Cyclin B1 Oncolytic CDK1/Cyclin B1
Dimethylsulfamoyl)phenylamino]- Inhibitors Drugs Inhibitors;
Inhibitors of 5-ethylpyrimidin-4- Signal Transduction
ylamino]ethyl]acetamide Pathways hydrochloride AZD-5438
4-(1-Isopropyl-2-methyl-1H- CDK1/Cyclin B1 Solid Tumors CDK1/Cyclin
B1 imidazol-5-yl)-N-[4- Inhibitors Therapy Inhibitors; CDK2/Cyclin
(methylsulfonyl)phenyl]pyrimidin- A Inhibitors; 2-amine CDK2/Cyclin
E Inhibitors CDK2/Cyclin A Inhibitors CDK2/Cyclin E Inhibitors
4-Butoxy-5-(phenylsulfinyl)-1H- CDK1/Cyclin B1 Oncolytic
CDK1/Cyclin B1 pyrazolo[3,4-b]pyridine Inhibitors Drugs Inhibitors;
CDK2/Cyclin E Inhibitors; CDK4/Cyclin D1 Inhibitors; Inhibitors of
Signal Transduction Pathways CDK2/Cyclin E Inhibitors CDK4/Cyclin
D1 Inhibitors 4-Butoxy-5-(phenylsulfonyl)-1H- CDK1/Cyclin B1
Oncolytic CDK1/Cyclin B1 pyrazolo[3,4-b]pyridine Inhibitors Drugs
Inhibitors; CDK2/Cyclin E Inhibitors; CDK4/Cyclin D1 Inhibitors;
Inhibitors of Signal Transduction Pathways CDK2/Cyclin E Inhibitors
CDK4/Cyclin D1 Inhibitors 2-[4-[5-[2,6-Dimethoxy-4-[5- CDK1/Cyclin
B1 Oncolytic Antimitotic Drugs; (3,4,5-trimethoxyphenyl)-4,5-
Inhibitors Drugs CDK1/Cyclin B1 dihydroisooxazol-3- Inhibitors;
Tubulin yl]phenoxy]pentyloxy]-3- polymerization
methoxyphenyl]-1,2,3,4- inhibitors tetrahydroquinazolin-4-one
Tubulin polymerization inhibitors Drug Name (CD, GN, Related BN)
Organization Condition Basic Patent Scytonemin GlaxoSmithKline;
Cancer; US University of Photosensitivity 2002022589; California,
Santa WO Barbara; 2001062900 U.S. Pat. No. 6,495,586; Issued Dec.
17, 2002, Expired on Jan. 17, 2007; U.S. Pat. No. 6,495,588, Issued
Dec. 17, 2002, Expired Jan. 17, 2007 Boehringer Cancer WO Ingelheim
2003032997 (Originator); AZD-5438 AstraZeneca Cancer, solid
(Originator); tumor Bristol-Myers Cancer WO Squibb 2001081348
(Originator); Bristol-Myers Cancer WO Squibb 2001081348
(Originator); CSIR (IN) Cancer WO (Originator); 2010058417
[0227] In some embodiments, the therapeutic regimen comprises
prescribing and/or administering an inhibitor of mdm2-induced
degradation of p53 to the subject. Such inhibitors include, but are
not limited to, Nutlin3, Nutlin3a, HL198C, benzodiazepines,
spiro-oxindoles, quillinois and RITA. Various inhibitors of the
p53-mdm2 interaaction are known in the art and are described in
Chene, Molec. Canc. Res., 2:20-28, 2004 and Vassilev et al., Trends
Molec. Med., 13:23-31, 2006, the disclosure of which are
incorporated herein by reference in their entireties. Such
inhibitors include chalcone derivative (compound B-1, Stoll et al.,
Biochem., 2:336-344, 2001), polycyclic compound (compound cyc-2,
Zhao et al., Cancer Lett., 2:69-77, 2002), chlorofusin (Duncan et
al., J. Am. Chem., 2:554-560, 2001), and various peptide inhibitors
(Ac-Gln-Glu-Thr-Phe-Ser-Asp-Leu-Trp-Lys-Lru-Leu-Pro-NH.sub.2-SEQ ID
NO: 7;
Ac-Met-Pro-Arg-Phe-Met-Asp-Tyr-Trp-Glu-Gly-Leu-Asn-NH.sub.2-SEQ ID
NO: 8; Ac-Phe-Met-Asp-Tyr-Trp-Glu-Gly-Leu-Asn-NH.sub.2-SEQ ID NO:
9; Ac-Glu-Thr-Phe-Aib-Asp-Aib-Trp-Lys-Aib-Leu-Aib-Glu NH.sub.2-SEQ
ID NO: 10; Ac-Phe-Met-Aib-Tyr-Trp-Glu-Ac3c-Leu-Asn-NH.sub.2-SEQ ID
NO: 11; Ac-Phe-Met-Aib-Pmp-Trp-Glu-Ac3c-Leu-Asn-NH.sub.2-SEQ ID NO:
12; and Ac-Phe-Met-Aib-Pmp-6ClTrp-Glu-Ac3c-Leu-Asn-NH.sub.2-SEQ ID
NO: 13)
[0228] The therapeutic regimen optionally comprises prescribing
and/or administering an inhibitor of B-RafV600E to the subject.
Exemplary inhibitors are described in Wenglowsky et al, ACS Med.
Chem., 2:342-347, 2011 and James et al., Mol. Cancer Ther., epub
Feb. 7, 2012, the disclosure of which are incorporated herein by
reference in their entireties and include, but are not limited to,
Vemurafenib (also known as PLX-4032, RG7204 or RO5185426, marketed
as Zelboraf.TM.) and CEP-32496).
[0229] In some embodiments, the therapeutic regimen comprises
prescribing a standard of care therapy to a mammalian subject in
whom elevated iASPP phosphorylation and/or elevated iASPP nuclear
localization is absent from the cancer cell.
[0230] V. Combination Therapy
[0231] Therapeutic compositions can be administered in
therapeutically effective dosages alone or in combination with
adjunct cancer therapy such as surgery, chemotherapy, radiotherapy,
thermotherapy, and laser therapy, and may provide a beneficial
effect, e.g. reducing tumor size, slowing rate of tumor growth,
inhibiting metastasis, or otherwise improving overall clinical
condition, without necessarily eradicating the cancer. Cytostatic
and cytotoxic agents that target the cancer cells are specifically
contemplated for combination therapy. Likewise, agents that target
angiogenesis or lymphangiogenesis are specifically contemplated for
combination therapy.
[0232] As used herein, a "chemotherapeutic agent" is a chemical
compound useful in the treatment of cancer. Examples of
chemotherapeutic agents include: alkylating agents such as thiotepa
and CYTOXAN.RTM. cyclosphosphamide; alkyl sulfonates such as
busulfan, improsulfan and piposulfan; aziridines such as benzodopa,
carboquone, meturedopa, and uredopa; ethylenimines and
methylamelamines including altretamine, triethylenemelamine,
trietylenephosphoramide, triethiylenethiophosphoramide and
tiimethylolomelamine; acetogenins (especially bullatacin and
bullatacinone); a camptothecin (including the synthetic analogue
topotecan); bryostatin; callystatin; CC-1065 (including its
adozelesin, carzelesin and bizelesin synthetic analogues);
cryptophycins (particularly cryptophycin 1 and cryptophycin 8);
dolastatin; duocarmycin (including the synthetic analogues, KW-2189
and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;
spongistatin; nitrogen mustards such as chlorambucil,
chlornaphazine, cholophosphamide, estramustine, ifosfamide,
mechlorethamine, mechlorethamine oxide hydrochloride, melphalan,
novembichin, phenesterine, prednimustine, trofosfamide, uracil
mustard; nitrosureas such as carmustine, chlorozotocin,
fotemustine, lomustine, nimustine, and ranimnustine; vinca
alkaloids; epipodophyllotoxins; antibiotics such as the enediyne
antibiotics (e.g., calicheamicin, especially calicheamicin gammall
and calicheamicin omegall; L-asparaginase; anthracenedione
substituted urea; methyl hydrazine derivatives; dynemicin,
including dynemicin A; bisphosphonates, such as clodronate; an
esperamicin; as well as neocarzinostatin chromophore and related
chromoprotein enediyne antiobiotic chromophores), aclacinomysins,
actinomycin, authramycin, azaserine, bleomycins, cactinomycin,
carabicin, carminomycin, carzinophilin, chromomycinis,
dactinomycin, daunorubicin, detorubicin,
6-diazo-5-oxo-L-norleucine, ADRIAMYCIN.RTM. doxorubicin (including
morpholino-doxorubicin, cyanomorpholino-doxorubicin,
2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin,
esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin
C, mycophenolic acid, nogalamycin, olivomycins, peplomycin,
potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin,
streptozocin, tubercidin, ubenimex, zinostatin, zorubicin;
anti-metabolites such as methotrexate and 5-fluorouracil (5-FU);
folic acid analogs such as denopterin, methotrexate, pteropterin,
trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine,
thiamiprine, thioguanine; pyrimidine analogs such as ancitabine,
azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine,
doxifluridine, enocitabine, floxuridine; androgens such as
calusterone, dromostanolone propionate, epitiostanol, mepitiostane,
testolactone; anti-adrenals such as aminoglutethimide, mitotane,
trilostane; folic acid replenisher such as frolinic acid;
aceglatone; aldophosphamide glycoside; aminolevulinic acid;
eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate;
defofamine; demecolcine; diaziquone; elfornithine; elliptinium
acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea;
lentinan; lonidainine; maytansinoids such as maytansine and
ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitiaerine;
pentostatin; phenamet; pirarubicin; losoxantione; podophyllinic
acid; 2-ethylhydrazide; procarbazine; PSK.RTM. polysaccharide
complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;
sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2
2''-trichlorotiiethylamine; trichothecenes (especially T-2 toxin,
verracurin A, roridin A and anguidine); urethan; vindesine;
dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;
gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa;
taxoids, e.g., TAXOL.RTM. paclitaxel (Bristol-Myers Squibb
Oncology, Princeton, N.J.), ABRAXANE.TM. Cremophor-free,
albumin-engineered nanoparticle formulation of paclitaxel (American
Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE.RTM.
docetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;
GEMZAR.RTM. gemcitabine; 6-thioguanine; mercaptopurine;
methotrexate; platinum coordination complexes such as cisplatin,
oxaliplatin and carboplatin; vinblastine; platinum; etoposide
(VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE.RTM.
vinorelbine; novantrone; teniposide; edatrexate; daunomycin;
aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11);
topoisomerase inhibitor RFS 2000; difluoromethylornithine (DFMO);
retinoids such as retinoic acid; capecitabine; leucovorin (LV);
irenotecan; adrenocortical suppressant; adrenocorticosteroids;
progestins; estrogens; androgens; gonadotropin-releasing hormone
analogs; and pharmaceutically acceptable salts, acids or
derivatives of any of the above. Also included in this definition
are anti-hormonal agents that act to regulate or inhibit hormone
action on tumors such as anti-estrogens and selective estrogen
receptor modulators (SERMs), including, for example, tamoxifen
(including NOLVADEX.RTM. tamoxifen), raloxifene, droloxifene,
4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone,
and FARESTON-toremifene; aromatase inhibitors that inhibit the
enzyme aromatase, which regulates estrogen production in the
adrenal glands, such as, for example, 4(5)-imidazoles,
aminoglutethimide, MEGASE.RTM. megestrol acetate, AROMASL.RTM.
exemestane, formestanie, fadrozole, RIVISOR.RTM. vorozole,
FEMARA.RTM. letrozole, and ARTMIDEX.RTM. anastrozole; and
anti-androgens such as flutamide, nilutamide, bicalutamide,
leuprolide, and goserelin; as well as troxacitabine (a
1,3-dioxolane nucleoside cytosine analog); antisense
oligonucleotides, particularly those which inhibit expression of
genes in signaling pathways implicated in abherant cell
proliferation, such as, for example, PKC-alpha, Ralf and H-Ras;
ribozymes such as a VEGF-A expression inhibitor (e.g.,
ANGIOZYME.RTM. ribozyme) and a HER2 expression inhibitor; vaccines
such as gene therapy vaccines, for example, ALLOVECTIN.RTM.
vaccine, LEUVECTIN.RTM. vaccine, and VAXID.RTM. vaccine;
PROLEUKIN.RTM. rJL-2; LURTOTECAN.RTM. topoisomerase 1 inhibitor;
ABARELLX.RTM. rmRH; and pharmaceutically acceptable salts, acids or
derivatives of any of the above.
[0233] A "growth inhibitory agent" as used herein refers to a
compound or composition which inhibits growth of a cell in vitro
and/or in vivo. Thus, the growth inhibitory agent may be one which
significantly reduces the percentage of cells in S phase. Examples
of growth inhibitory agents include agents that block cell cycle
progression (at a place other than S phase), such as agents that
induce G1 arrest and M-phase arrest. Classical M-phase blockers
include the vincas (vincristine and vinblastine), TAXOL.RTM., and
topo II inhibitors such as doxorubicin, epirubicin, daunorubicin,
etoposide, and bleomycin. Those agents that arrest G1 also spill
over into S-phase arrest, for example, DNA alkylating agents such
as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin,
methotrexate, 5-fluorouracil, and ara-C.
[0234] Cytokines that are effective in inhibiting tumor metastasis
are also contemplated for use in the combination therapy. Such
cytokines, lymphokines, or other hematopoietic factors include, but
are not limited to, M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4,
IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14,
IL-15, IL-16, IL-17, IL-18, IFN, TNF.alpha., TNF1, TNF2, G-CSF,
Meg-CSF, GM-CSF, thrombopoietin, stem cell factor, and
erythropoietin.
[0235] The treatment methods described herein optionally include
monitoring the effect of the therapeutic composition on the tumor.
For example, the size of the tumor can be determined, as can the
presence of metastases. These compositions would be provided in a
combined amount effective to kill or inhibit proliferation of the
cancer by killing and/or inhibiting the proliferation of the cancer
cells. This process may involve contacting the cells with a agent
that inhibits phosphorylation of iASPP and a further agent(s) at
the same time. This may be achieved by contacting the cell with a
single composition or pharmacological formulation that includes
both agents, or by contacting the cell with two distinct
compositions or formulations, at the same time, wherein one
composition includes an agent that inhibits phosphorylation of
iASPP or an iASPP inhibitor peptide and the other includes a second
agent.
[0236] Combination therapy with an agent described herein may be
achieved by administering to a subject a single composition or
pharmacological formulation that includes the cdk1 inhibitor or
inhibitor peptides and the one or more additional agents, or by
administering to the subject two (or more) distinct compositions or
formulations, at the same time, wherein one composition includes a
cdk1 inhibitor or inhibitor peptides and the other includes a
second agent.
[0237] Alternatively, the combination therapy employing a cdk1
inhibitor described herein may precede or follow the second agent
treatment by intervals ranging from minutes to weeks. In
embodiments where the second agent and the cdk1 inhibitor are
administered separately, one would generally ensure that a
significant period of time did not expire between the times of each
delivery, such that the agent and the cdk1 inhibitor would still be
able to exert an advantageously combined effect. In such instances,
it is contemplated that one would administer both modalities within
about 12-24 hours of each other and, more preferably, within about
6-12 hours of each other, with a delay time of only about 12 hours
being most preferred. In some situations, it may be desirable to
extend the time period for treatment significantly, however, where
several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5,
6, 7 or 8) lapse between the respective administrations. Repeated
treatments with one or both agents is specifically
contemplated.
[0238] VI. Pharmaceutical Compositions and Routes of
Administration
[0239] Purified protein, antibodies, antagonists, or inhibitors may
all be used as pharmaceutical compositions. Delivery of specific
molecules for therapeutic purposes in this invention is further
described below.
[0240] The active compositions of the present invention include
classic pharmaceutical preparations. Administration of these
compositions according to the present invention will be via any
common route so long as the target tissue is available via that
route. The pharmaceutical compositions may be introduced into the
subject by any conventional method, e.g., by intravenous,
intradermal, intramusclar, intramammary, intraperitoneal,
intrathecal, intraocular, retrobulbar, intrapulmonary (e.g., term
release); by oral, sublingual, nasal, anal, vaginal, or transdermal
delivery, or by surgical implantation at a particular site, e.g.,
embedded under the splenic capsule, brain, or in the cornea. The
treatment may consist of a single dose or a plurality of doses over
a period of time.
[0241] For all protein-based therapeutics described herein (e.g.,
antibodies) administration by the delivery of gene expression
constructs are contemplated as one embodiment. Any suitable vector
may be used to introduce a polynucleotide that encodes a
protein-based therapeutic described herein, into the host.
Exemplary vectors that have been described in the literature
include replication deficient retroviral vectors, including but not
limited to lentivirus vectors [Kim et al., J. Virol., 72(1):
811-816 (1998); Kingsman & Johnson, Scrip Magazine, October,
1998, pp. 43 46.]; adeno-associated viral (AAV) vectors [U.S. Pat.
No. 5,474,935; U.S. Pat. No. 5,139,941; U.S. Pat. No. 5,622,856;
U.S. Pat. No. 5,658,776; U.S. Pat. No. 5,773,289; U.S. Pat. No.
5,789,390; U.S. Pat. No. 5,834,441; U.S. Pat. No. 5,863,541; U.S.
Pat. No. 5,851,521; U.S. Pat. No. 5,252,479; Gnatenko et al., J.
Invest. Med., 45: 87 98 (1997)]; adenoviral (AV) vectors [See,
e.g., U.S. Pat. No. 5,792,453; U.S. Pat. No. 5,824,544; U.S. Pat.
No. 5,707,618; U.S. Pat. No. 5,693,509; U.S. Pat. No. 5,670,488;
U.S. Pat. No. 5,585,362; Quantin et al., Proc. Natl. Acad. Sci.
USA, 89: 2581 2584 (1992); Stratford Perricadet et al., J. Clin.
Invest., 90: 626 630 (1992); and Rosenfeld et al., Cell, 68: 143
155 (1992)]; an adenoviral adenoassociated viral chimeric (see for
example, U.S. Pat. No. 5,856,152) or a vaccinia viral or a
herpesviral (see for example, U.S. Pat. No. 5,879,934; U.S. Pat.
No. 5,849,571; U.S. Pat. No. 5,830,727; U.S. Pat. No. 5,661,033;
U.S. Pat. No. 5,328,688; Lipofectin mediated gene transfer (BRL);
liposomal vectors [See, e.g., U.S. Pat. No. 5,631,237 (Liposomes
comprising Sendai virus proteins)]; and combinations thereof. All
of the foregoing documents are incorporated herein by reference in
their entirety. Replication deficient adenoviral vectors constitute
a preferred embodiment.
[0242] The active compounds may be prepared for administration as
solutions of free base or pharmacologically acceptable salts in
water suitably mixed with a surfactant, such as
hydroxypropylcellulose. Dispersions also can be prepared in
glycerol, liquid polyethylene glycols, and mixtures thereof and in
oils. Under ordinary conditions of storage and use, these
preparations contain a preservative to prevent the growth of
microorganisms.
[0243] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions or dispersions and sterile powders for
the extemporaneous preparation of sterile injectable solutions or
dispersions. In all cases the form must be sterile and must be
fluid to the extent that easy syringability exists. It must be
stable under the conditions of manufacture and storage and must be
preserved against the contaminating action of microorganisms, such
as bacteria and fungi. The carrier can be a solvent or dispersion
medium containing, for example, water, ethanol, polyol (for
example, glycerol, propylene glycol, and liquid polyethylene
glycol, and the like), suitable mixtures thereof, and vegetable
oils. The proper fluidity can be maintained, for example, by the
use of a coating, such as lecithin, by the maintenance of the
required particle size in the case of dispersion and by the use of
surfactants. The prevention of the action of microorganisms can be
brought about by various antibacterial and antifungal agents, for
example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal,
and the like. In many cases, it will be preferable to include
isotonic agents, for example, sugars or sodium chloride. Prolonged
absorption of the injectable compositions can be brought about by
the use in the compositions of agents delaying absorption, for
example, aluminum monostearate and gelatin.
[0244] Sterile injectable solutions are prepared by incorporating
the active compounds in the required amount in the appropriate
solvent with various of the other ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterilized
active ingredients into a sterile vehicle which contains the basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum-drying and freeze-drying techniques which
yield a powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof.
[0245] As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents and the like. The use of such media and agents for
pharmaceutical active substances is well known in the art. Except
insofar as any conventional media or agent is incompatible with the
active ingredient, its use in the therapeutic compositions is
contemplated. Supplementary active ingredients also can be
incorporated into the compositions.
[0246] For oral administration the active compositions may be
incorporated with excipients and used in the form of non-ingestible
mouthwashes and dentifrices. A mouthwash may be prepared
incorporating the active ingredient in the required amount in an
appropriate solvent, such as a sodium borate solution (Dobell's
Solution). Alternatively, the active ingredient may be incorporated
into an antiseptic wash containing sodium borate, glycerin and
potassium bicarbonate. The active ingredient may also be dispersed
in dentifrices, including: gels, pastes, powders and slurries. The
active ingredient may be added in a therapeutically effective
amount to a paste dentifrice that may include water, binders,
abrasives, flavoring agents, foaming agents, and humectants.
[0247] The compositions of the present invention may be formulated
in a neutral or salt form. Pharmaceutically-acceptable salts
include the acid addition salts (formed with the free amino groups
of the protein) and which are formed with inorganic acids such as,
for example, hydrochloric or phosphoric acids, or such organic
acids as acetic, oxalic, tartaric, mandelic, and the like. Salts
formed with the free carboxyl groups also can be derived from
inorganic bases such as, for example, sodium, potassium, ammonium,
calcium, or ferric hydroxides, and such organic bases as
isopropylamine, trimethylamine, histidine, procaine and the
like.
[0248] The compositions described herein may be formulated in a
neutral or salt form. Pharmaceutically-acceptable salts include the
acid addition salts (formed with the free amino groups of the
protein) and which are formed with inorganic acids such as, for
example, hydrochloric or phosphoric acids, or such organic acids as
acetic, oxalic, tartaric, mandelic, and the like. Salts formed with
the free carboxyl groups also can be derived from inorganic bases
such as, for example, sodium, potassium, ammonium, calcium, or
ferric hydroxides, and such organic bases as isopropylamine,
trimethylamine, histidine, procaine and the like.
[0249] Upon formulation, solutions will be administered in a manner
compatible with the dosage formulation and in such amount as is
therapeutically effective. The formulations are easily administered
in a variety of dosage forms such as injectable solutions, drug
release capsules and the like. For parenteral administration in an
aqueous solution, for example, the solution should be suitably
buffered if necessary and the liquid diluent first rendered
isotonic with sufficient saline or glucose. These particular
aqueous solutions are especially suitable for intravenous,
intramuscular, subcutaneous and intraperitoneal administration.
[0250] In the clinical setting an "effective amount" is an amount
sufficient to effect beneficial or desired clinical results. An
effective amount can be administered in one or more doses. In terms
of treatment, an "effective amount" of a therapeutic agent
described herein is an amount that results in amelioration of
symptoms or a prolongation of survival in a subject. The effective
amount is generally determined by the physician on a case-by-case
basis and is within the skill of one in the art. Several factors
are typically taken into account when determining, an appropriate
dosage. These factors include age, sex and weight of the patient,
the condition being treated, the severity of the condition.
Determination of the effective amounts is well within the
capability of those skilled in the art, especially in light of the
detailed disclosure provided herein. For any therapeutic agent used
in a method described herein, the therapeutically effective dose
can be estimated initially from cell culture assays. For example, a
dose can be formulated in animal models to achieve a circulating
concentration range that includes the IC.sub.50 as determined in
cell culture (i.e., the concentration of the test compound which
achieves a half-maximal inhibition of the C-proteinase activity).
Such information can be used to more accurately determine useful
doses in humans.
[0251] Toxicity and therapeutic efficacy of such compounds can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD.sub.50 (the
dose lethal to 50% of the population) and the ED50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio between LD50 and ED50. Compounds
which exhibit high therapeutic indices are preferred. The data
obtained from these cell culture assays and animal studies can be
used in formulating a range of dosage for use in human. The dosage
of such compounds lies preferably within a range of circulating
concentrations that include the ED.sub.50 with little or no
toxicity. The dosage may vary within this range depending upon the
dosage form employed and the route of administration utilized. The
exact formulation, route of administration and dosage can be chosen
by the individual physician in view of the patient's condition.
See, e.g., Fingl et al., 1975, in "The Pharmacological Basis of
Therapeutics," Ch. 1 p. 1.Dosage amount and interval may be
adjusted individually to provide plasma levels of the active moiety
which are sufficient to maintain the C-proteinase inhibiting
effects, or minimal effective concentration (MEC). The MEC will
vary for each compound but can be estimated from in vitro data; for
example, the concentration necessary to achieve 50-90% inhibition
of the C-proteinase using the assays described herein. Dosages
necessary to achieve the MEC will depend on individual
characteristics and route of administration. However, HPLC assays
or bioassays can be used to determine plasma concentrations.
[0252] Dosage intervals can also be determined using MEC value.
Compounds should be administered using a regimen which maintains
plasma levels above the MEC for 10-90% of the time, preferably
between 30-90% and most preferably between 50-90%. In cases of
local administration or selective uptake, the effective local
concentration of the drug may not be related to plasma
concentration. Refinement of the calculations necessary to
determine the appropriate treatment dose is routinely made by those
of ordinary skill in the art without undue experimentation,
especially in light of the dosage information and assays disclosed
herein as well as the pharmacokinetic data observed in animals or
human clinical trials. As studies are conducted, further
information will emerge regarding appropriate dosage levels and
duration of treatment for specific diseases and conditions.
[0253] In a preferred embodiment, the present invention is directed
at treatment of a cancer selected from the group consisting of
melanoma, prostate cancer, head cancer and neck cancer, wherein the
cancer expresses an elevated level of phosphorylated iASPP. A
variety of different routes of administration are contemplated. For
example, in the case of a tumor, the discrete tumor mass may be
injected with a therapeutic agent described herein. The injections
may be single or multiple; where multiple, injections are made at
about 1 cm spacings across the accessible surface of the tumor.
Further, systemic injection may be preferred.
[0254] The pharmaceutical compositions and treatment methods of the
invention may be useful in fields of human medicine and veterinary
medicine. Thus the subject to be treated may be a mammal,
preferably human or other animal. For veterinary purposes, subjects
include for example, farm animals including cows, sheep, pigs,
horses and goats, companion animals such as dogs and cats, exotic
and/or zoo animals, laboratory animals including mice rats,
rabbits, guinea pigs and hamsters; and poultry such as chickens,
turkey ducks and geese.
[0255] The compositions may, if desired, be presented in a pack or
dispenser device which may contain one or more unit dosage forms
containing the active ingredient. The pack may, for example,
comprise metal or plastic foil, such as a blister pack. The pack or
dispenser device may be accompanied by instructions for
administration. Compositions comprising a compound of the invention
formulated in a compatible pharmaceutical carrier may also be
prepared, placed in an appropriate container, and labeled for
treatment of an indicated condition.
[0256] VII. Kits
[0257] In another embodiment, kits are provided which contain the
necessary reagents to carry out the assays or therapies of the
present invention. In some variations, reagents are packaged
together but not in admixture. In one embodiment, the invention
provides a compartment kit to receive, in close confinement, one or
more containers which comprises: (a) a first container comprising
an antibody that specifically binds iASPP polypeptide and
preferentially binds to iASPP having a phosphorylated serine
residue compared to binding without the phosphorylated serine,
wherein the serine residue corresponds to a serine selected from
the group consisting of Ser84 and Ser113 of SEQ ID NO: 2;
optionally (b) a second container comprising an antibody that
specifically binds iASPP polypeptide, but does not bind to iASPP
having the phosphorylated serine residue; and (c) one or more other
containers comprising one or more of the following: wash reagents,
reagents capable of detecting presence of a bound antibody.
[0258] In another embodiments, the invention provides a compartment
kit to receive, in close confinement, one or more containers which
comprises (a) a first container comprising an antibody that
specifically binds iASPP; optionally (b) a second container
comprising an antibody that specifically binds to iASPP having a
phosphorylated serine residue compared to binding without the
phosphorylated serine, wherein the serine residue corresponds to a
serine selected from the group consisting of Ser84 and Ser113 of
SEQ ID NO: 2; and (c) one or more other containers comprising one
or more of the following: wash reagents, reagents capable of
detecting presence of a bound antibody.
[0259] In further embodiments, the invention provides a compartment
kit to receive, in close confinement, one or more containers which
comprises (a) a first container comprising an antibody that
specifically binds iASPP; optionally (b) a second container
comprising an antibody that specifically binds iASPP polypeptide,
but does not bind to iASPP having the phosphorylated serine residue
and (c) one or more other containers comprising one or more of the
following: wash reagents, reagents capable of detecting presence of
a bound antibody.
[0260] The invention also provides, in some embodiments, a
compartment kit to receive, in close confinement, one or more
containers which comprises (a) a first container comprising an
antibody that specifically binds iASPP; (b) a second container
comprising an antibody that specifically binds to iASPP having a
phosphorylated serine residue compared to binding without the
phosphorylated serine, wherein the serine residue corresponds to a
serine selected from the group consisting of Ser84 and Ser113 of
SEQ ID NO: 2; and optionally (c) a third container comprising an
antibody that specifically binds iASPP polypeptide, but does not
bind to iASPP having the phosphorylated serine residue and (d) one
or more other containers comprising one or more of the following:
wash reagents, reagents capable of detecting presence of a bound
antibody.
[0261] In some embodiments, described herein is a compartment kit
to receive, in close confinement (such as a single box or package,
one or more containers which comprises (a) a first container
comprising a first antibody that binds an iASPP polypeptide, (b) a
second container comprising a second antibody that binds a region
of iASPP that includes Ser84 or Ser113 of iASPP (SEQ ID NO: 2) and
selectively binds iASPP when said serine in said epitope is
unphosphorylated, and wherein the second binding partner optionally
comprises a detectable label; (c) one or more other containers
comprising one or more of the following: wash reagents, reagents
capable of detecting presence of a bound antibody; and optionally
(d) a third container comprising a nucleus specific marker.
[0262] In another embodiment, the invention provides a compartment
kit to receive, in close confinement, one or more containers which
comprises (a) a first container comprising an antibody that
specifically binds iASPP; optionally (b) a second container
comprising an antibody that specifically binds to iASPP having a
phosphorylated serine residue compared to binding without the
phosphorylated serine, wherein the second antibody comprises a
detectable label; (c) one or more other containers comprising one
or more of the following: wash reagents, reagents capable of
detecting presence of a bound antibody; and optionally (d) a third
container comprising a nucleus specific marker.
[0263] In detail, a compartment kit includes any kit in which
reagents are contained in separate containers. Such containers
include small glass containers, plastic containers or strips of
plastic or paper. Such containers allow one to efficiently transfer
reagents from one compartment to another compartment such that the
samples and reagents are not cross-contaminated, and the agents or
solutions of each container can be added in a quantitative fashion
from one compartment to another. Such containers will include a
container which will accept the test sample, a container which
contains the antibody or antibodies used in the assay, containers
which contain wash reagents (such as phosphate-buffered saline,
Tris buffers, and the like), and containers which contain the
reagents used to detect the bound antibody or probe. Types of
detection reagents include labeled nucleic acid probes, labeled
secondary antibodies, or in the alternative, if the primary
antibody is labeled, the enzymatic, or antibody binding reagents
which are capable of reacting with the labeled antibody. One
skilled in the art will readily recognize that the disclosed
antibodies of the present invention can be readily incorporated
into one of the established kit formats which are well known in the
art.
[0264] In further detail, kits for use in measuring phosphorylated
iASPP in a sample can include a first antibody or functional
fragment thereof which binds to iASPP (both phosphorylated and
unphosphorylated forms) or portion of this iASPP, a second antibody
or functional fragment thereof that specifically binds
phosphorylated iASPP or a portion of the phosphorylated iASPP as
well as one or more ancillary reagents suitable for detecting the
presence of a complex between the antibodies (or fragment) and
iASPP or portion thereof. The antibody compositions can be provided
in lyophilized form, either alone or in combination with additional
antibodies specific for other epitopes. The antibodies, which can
be labeled or unlabeled, can be included in the kits with adjunct
ingredients. For example, the antibodies can be provided as a
lyophilized mixture with the adjunct ingredients, or the adjunct
ingredients can be separately provided for combination by the user.
Generally these adjunct materials will be present in less than
about 5% weight based on the amount of active antibody, and usually
will be present in a total amount of at least about 0.001% weight
based on antibody concentration. Where a second antibody capable of
binding to the monoclonal antibody is employed, such antibody can
be provided in the kit, for instance in a separate vial or
container. The second antibody, if present, is typically labeled,
and can be formulated in an analogous manner with the antibody
formulations described above.
[0265] In some embodiments, the kits described herein are packaged
with nuclear marker reagents.
[0266] VI. Diagnostic Systems
[0267] Another aspect of the invention is a system that is capable
of carrying out a part or all of a method of the invention, or
carrying out a variation of a method of the invention as described
herein in greater detail. Exemplary systems include, as one or more
components, computing systems, environments, and/or configurations
that may be suitable for use with the methods and include, but are
not limited to, personal computers, server computers, hand-held or
laptop devices, multiprocessor systems, microprocessor-based
systems, set top boxes, programmable consumer electronics, network
PCs, minicomputers, mainframe computers, distributed computing
environments that include any of the above systems or devices, and
the like. In some variations, a system of the invention includes
one or more machines used for analysis of biological material
(e.g., genetic material), as described herein. In some variations,
this analysis of the biological material involves a chemical
analysis and/or a nucleic acid amplification.
[0268] With reference to FIG. 15, an exemplary system of the
invention, which may be used to implement one or more steps of
methods of the invention, includes a computing device in the form
of a computer 110. Components shown in dashed outline are not
technically part of the computer 110, but are used to illustrate
the exemplary embodiment of FIG. 15. Components of computer 110 may
include, but are not limited to, a processor 120, a system memory
130, a memory/graphics interface 121, also known as a Northbridge
chip, and an I/O interface 122, also known as a Southbridge chip.
The system memory 130 and a graphics processor 190 may be coupled
to the memory/graphics interface 121. A monitor 191 or other
graphic output device may be coupled to the graphics processor
190.
[0269] A series of system busses may couple various system
components including a high speed system bus 123 between the
processor 120, the memory/graphics interface 121 and the I/O
interface 122, a front-side bus 124 between the memory/graphics
interface 121 and the system memory 130, and an advanced graphics
processing (AGP) bus 125 between the memory/graphics interface 121
and the graphics processor 190. The system bus 123 may be any of
several types of bus structures including, by way of example, and
not limitation, such architectures include Industry Standard
Architecture (ISA) bus, Micro Channel Architecture (MCA) bus and
Enhanced ISA (EISA) bus. As system architectures evolve, other bus
architectures and chip sets may be used but often generally follow
this pattern. For example, companies such as Intel and AMD support
the Intel Hub Architecture (IHA) and the Hypertransport.TM.
architecture, respectively.
[0270] The computer 110 typically includes a variety of
computer-readable media. Computer-readable media can be any
available media that can be accessed by computer 110 and includes
both volatile and nonvolatile media, removable and non-removable
media. By way of example, and not limitation, computer readable
media may comprise computer storage media. Computer storage media
includes both volatile and nonvolatile, removable and non-removable
media implemented in any method or technology for storage of
information such as computer readable instructions, data
structures, program modules or other data. Computer storage media
includes, but is not limited to, RAM, ROM, EEPROM, flash memory or
other memory technology, CD-ROM, digital versatile disks (DVD) or
other optical disk storage, magnetic cassettes, magnetic tape,
magnetic disk storage or other magnetic storage devices, or any
other physical medium which can be used to store the desired
information and which can accessed by computer 110.
[0271] The system memory 130 includes computer storage media in the
form of volatile and/or nonvolatile memory such as read only memory
(ROM) 131 and random access memory (RAM) 132. The system ROM 131
may contain permanent system data 143, such as identifying and
manufacturing information. In some embodiments, a basic
input/output system (BIOS) may also be stored in system ROM 131.
RAM 132 typically contains data and/or program modules that are
immediately accessible to and/or presently being operated on by
processor 120. By way of example, and not limitation, FIG. 15
illustrates operating system 134, application programs 135, other
program modules 136, and program data 137.
[0272] The I/O interface 122 may couple the system bus 123 with a
number of other busses 126, 127 and 128 that couple a variety of
internal and external devices to the computer 110. A serial
peripheral interface (SPI) bus 126 may connect to a basic
input/output system (BIOS) memory 133 containing the basic routines
that help to transfer information between elements within computer
110, such as during start-up.
[0273] A super input/output chip 160 may be used to connect to a
number of `legacy` peripherals, such as floppy disk 152,
keyboard/mouse 162, and printer 196, as examples. The super I/O
chip 160 may be connected to the I/O interface 122 with a bus 127,
such as a low pin count (LPC) bus, in some embodiments. Various
embodiments of the super I/O chip 160 are widely available in the
commercial marketplace.
[0274] In one embodiment, bus 128 may be a Peripheral Component
Interconnect (PCI) bus, or a variation thereof, may be used to
connect higher speed peripherals to the I/O interface 122. A PCI
bus may also be known as a Mezzanine bus. Variations of the PCI bus
include the Peripheral Component Interconnect-Express (PCI-E) and
the Peripheral Component Interconnect-Extended (PCI-X) busses, the
former having a serial interface and the latter being a backward
compatible parallel interface. In other embodiments, bus 128 may be
an advanced technology attachment (ATA) bus, in the form of a
serial ATA bus (SATA) or parallel ATA (PATA).
[0275] The computer 110 may also include other
removable/non-removable, volatile/nonvolatile computer storage
media. By way of example only, FIG. 15 illustrates a hard disk
drive 140 that reads from or writes to non-removable, nonvolatile
magnetic media. The hard disk drive 140 may be a conventional hard
disk drive.
[0276] Removable media, such as a universal serial bus (USB) memory
153, firewire (IEEE 1394), or CD/DVD drive 156 may be connected to
the PCI bus 128 directly or through an interface 150. A storage
media 154 may coupled through interface 150. Other
removable/non-removable, volatile/nonvolatile computer storage
media that can be used in the exemplary operating environment
include, but are not limited to, magnetic tape cassettes, flash
memory cards, digital versatile disks, digital video tape, solid
state RAM, solid state ROM, and the like.
[0277] The drives and their associated computer storage media
discussed above and illustrated in FIG. 15, provide storage of
computer readable instructions, data structures, program modules
and other data for the computer 110. In FIG. 15, for example, hard
disk drive 140 is illustrated as storing operating system 144,
application programs 145, other program modules 146, and program
data 147. Note that these components can either be the same as or
different from operating system 134, application programs 135,
other program modules 136, and program data 137. Operating system
144, application programs 145, other program modules 146, and
program data 147 are given different numbers here to illustrate
that, at a minimum, they are different copies. A user may enter
commands and information into the computer 20 through input devices
such as a mouse/keyboard 162 or other input device combination.
Other input devices (not shown) may include a microphone, joystick,
game pad, satellite dish, scanner, or the like. These and other
input devices are often connected to the processor 120 through one
of the I/O interface busses, such as the SPI 126, the LPC 127, or
the PCI 128, but other busses may be used. In some embodiments,
other devices may be coupled to parallel ports, infrared
interfaces, game ports, and the like (not depicted), via the super
I/O chip 160.
[0278] The computer 110 may operate in a networked environment
using logical connections to one or more remote computers, such as
a remote computer 180 via a network interface controller (NIC) 170.
The remote computer 180 may be a personal computer, a server, a
router, a network PC, a peer device or other common network node,
and typically includes many or all of the elements described above
relative to the computer 110. The logical connection between the
NIC 170 and the remote computer 180 depicted in FIG. 15 may include
a local area network (LAN), a wide area network (WAN), or both, but
may also include other networks. Such networking environments are
commonplace in offices, enterprise-wide computer networks,
intranets, and the Internet. The remote computer 180 may also
represent a web server supporting interactive sessions with the
computer 110, or in the specific case of location-based
applications may be a location server or an application server.
[0279] In some embodiments, the network interface may use a modem
(not depicted) when a broadband connection is not available or is
not used. It will be appreciated that the network connection shown
is exemplary and other means of establishing a communications link
between the computers may be used.
[0280] In some variations, the invention is a system for
identifying susceptibility to a cancer in a human subject. For
example, in one variation, the system includes tools for performing
at least one step, preferably two or more steps, and in some
aspects all steps of a method of the invention, where the tools are
operably linked to each other. Operable linkage describes a linkage
through which components can function with each other to perform
their purpose.
[0281] In some variations, a system of the invention is a system
for identifying metastatic potential of a cancer in a human
subject, the system comprising:
[0282] (a) at least one processor;
[0283] (b) at least one computer-readable medium;
[0284] (c) a database operatively coupled to a computer-readable
medium of the system and containing population information
correlating the phosphorylation state of iASPP in cancer and
metastasis data in a population of humans;
[0285] (d) a measurement tool that receives an input about the
human subject and generates information from the input about the
iASPP phosphorylation in a cancer cell from the human subject;
and
[0286] (e) an analysis tool or routine that:
[0287] (i) is operatively coupled to the database and the
measurement tool,
[0288] (ii) is stored on a computer-readable medium of the
system,
[0289] (iii) is adapted to be executed on a processor of the
system, to compare the information about the human subject with the
population information in the susceptibility database and generate
a conclusion with respect to susceptibility to metastatic potential
of the cancer in the human subject.
[0290] Exemplary processors (processing units) include all variety
of microprocessors and other processing units used in computing
devices. Exemplary computer-readable media are described above.
When two or more components of the system involve a processor or a
computer-readable medium, the system generally can be created where
a single processor and/or computer readable medium is dedicated to
a single component of the system; or where two or more functions
share a single processor and/or share a single computer readable
medium, such that the system contains as few as one processor
and/or one computer readable medium. In some variations, it is
advantageous to use multiple processors or media, for example,
where it is convenient to have components of the system at
different locations. For instance, some components of a system may
be located at a testing laboratory dedicated to laboratory or data
analysis, whereas other components, including components (optional)
for supplying input information or obtaining an output
communication, may be located at a medical treatment or counseling
facility (e.g., doctor's office, health clinic, HMO, pharmacist,
geneticist, hospital) and/or at the home or business of the human
subject (patient) for whom the testing service is performed.
[0291] Referring to FIG. 16 an exemplary system includes a database
208 that is operatively coupled to a computer-readable medium of
the system and that contains population information correlating the
phosphorylation state of iASPP and metastatic potential of a cancer
in a population of humans. For example, for a statistically
meaningful number of cancer subjects, information is collected as
to whether the cancer was metastatic and as to whether (or what
percentage) of the cells have iASPP that was phosphorylated or
localized to the nucleus.
[0292] In a simple variation, the database contains 208 data
relating to the level of phosphorylated iASPP observed in cancer
cells of a population of humans with the cancer and information
about the metastatic behavior of the cancer. Such data provides an
indication as to the potential of developing metastatic cancer for
a human subject that is identified as expressing phosphorylated
iASPP. In another variation, the database optionally includes
similar data with respect to a level of iASPP within the nucleus of
a cancer cell compared to a level of iASPP in the cytoplasm of the
cancer cell. In still another variation, the database includes
additional quantitative personal, medical, or genetic information
about the individuals in the database with non-metastatic,
metastatic, or aggressively metastatic cancer (or control
individuals free of the cancer). As the database becomes more
populated with patient data, it become a more powerful statistical
tool for comparing an input with respect to a subject and making a
prediction as to metastatic potential. Such information includes,
but is not limited to, information about parameters such as age,
sex, ethnicity, race, medical history, weight, diabetes status,
blood pressure, family history of the cancer, smoking history, and
alcohol use in humans and impact of the at least one parameter on
metastatic potential of the cancer. These more robust databases can
be used by an analysis routine 210 to calculate a combined score
with respect to potential for a cancer to metastasize.
[0293] In addition to the database 208, the system further includes
a measurement tool 206 programmed to receive an input 204 from or
about the human subject and generate an output that contains
information about the phosphorylation state of iASPP. (The input
204 is not part of the system per se but is illustrated in the
schematic FIG. 16.) Thus, the input 204 will contain a specimen or
contain data from which a level of phosphorylated iASPP in a cancer
cell (or a level of iASPP within the nucleus of a cancer cell) can
be directly read, or analytically determined.
[0294] In another variation, the input 204 from the human subject
contains data that is unannotated or insufficiently annotated with
respect to iASPP, requiring analysis by the measurement tool 206.
For example, the input can be raw data measurements from
experiments designed to evaluate phosphorylation or localization of
iASPP. In such variations, the measurement tool 206 comprises a
tool, preferably stored on a computer-readable medium of the system
and adapted to be executed on a processor of the system, to receive
a data input about a subject and determine information about the
iASPP phosphorylation state from the data. For example, the
measurement tool 206 contains instructions, preferably executable
on a processor of the system, for analyzing the unannotated input
data and determining the level of iASPP phosphorylation of interest
in the human subject.
[0295] In yet another variation, the input 204 from the human
subject comprises a biological sample, such as a fluid (e.g.,
blood) or tissue sample, that contains one or more cancer cells or
iASPP protein that can be analyzed to determine the iASPP
phosphorylation state. In this variation, an exemplary measurement
tool 206 includes laboratory equipment for processing and analyzing
the sample to determine the iASPP phosphorylation state in cancer
cells of the human subject.
[0296] In some variations the measurement tool 206 includes:
immunoassay reagents for measuring iASPP and measuring iASPP
phosphorylation from cells in the biological sample; and an
analysis tool stored on a computer-readable medium of the system
and adapted to be executed on a processor of the system, to
determine the level of phosphorylated iASPP based on the
immunoassay data.
[0297] In some variations, the measurement tool 206 further
includes additional equipment and/or chemical reagents for
processing the biological sample to purify iASPP from cells in a
sample for further analysis using immunoassays, size separation
tools, or other analytical equipment.
[0298] The exemplary system further includes an analysis tool or
routine 210 that: is operatively coupled to the database 208 and
operatively coupled to the measurement tool 206, is stored on a
computer-readable medium of the system, is adapted to be executed
on a processor of the system to compare the information about the
human subject with the population information in the database 208
and generate a conclusion with respect to metastatic potential of
cancer in the human subject. In simple terms, the analysis tool 210
looks at the level of phosphorylated iASPP identified by the
measurement tool 206 for the human subject, and compares this
information to the database 208, to determine metastatic potential
of a cancer in the subject. The susceptibility can be based on the
single parameter (the level of phosphorylated iASPP), or can
involve a calculation based on other genetic and non-genetic data,
as described above, that is collected and included as part of the
input 204 from the human subject, and that also is stored in the
database 208 with respect to a population of other humans.
Generally speaking, each parameter of interest is weighted to
provide a conclusion with respect to metastatic potential of the
cancer. Such a conclusion is expressed in the conclusion in any
statistically useful form, for example, as an odds ratio or a
probability that the subject's cancer is a metastatic form of the
cancer (or has high potential for metastasis).
[0299] In some variations, the system as just described further
includes a communication tool 212. For example, the communication
tool is operatively connected to the analysis routine 210 and
comprises a routine stored on a computer-readable medium of the
system and adapted to be executed on a processor of the system, to:
generate a communication containing the conclusion; and to transmit
the communication to the human subject 200 or the medical
practitioner 202, and/or enable the subject or medical practitioner
to access the communication. (The subject and medical practitioner
are depicted in the schematic FIG. 17, but are not part of the
system per se, though they may be considered users of the system.
The communication tool 212 provides an interface for communicating
to the subject, or to a medical practitioner for the subject (e.g.,
doctor, nurse, genetic counselor), the conclusion generated by the
analysis tool 210 with respect to metastatic potential of a cancer
in the subject. Usually, if the communication is obtained by or
delivered to the medical practitioner 202, the medical practitioner
will share the communication with the human subject 200 and/or
counsel the human subject about the medical significance of the
communication. In some variations, the communication is provided in
a tangible form, such as a printed report or report stored on a
computer readable medium such as a flash drive or optical disk. In
some variations, the communication is provided electronically with
an output that is visible on a video display or audio output (e.g.,
speaker). In some variations, the communication is transmitted to
the subject or the medical practitioner, e.g., electronically or
through the mail. In some variations, the system is designed to
permit the subject or medical practitioner to access the
communication, e.g., by telephone or computer. For instance, the
system may include software residing on a memory and executed by a
processor of a computer used by the human subject or the medical
practitioner, with which the subject or practitioner can access the
communication, preferably securely, over the internet or other
network connection. In some variations of the system, this computer
will be located remotely from other components of the system, e.g.,
at a location of the human subject's or medical practitioner's
choosing.
[0300] In some variations, the system as described (including
embodiments with or without the communication tool) further
includes components that add a treatment or prophylaxis utility to
the system. For instance, value is added to a determination of
metastatic potential of a cancer when a medical practitioner can
prescribe or administer a standard of care that can reduce the
metastatis potential of the cancer; and/or delay onset of the
metastasis to facilitate early treatment and is most curable.
Exemplary medicinal and surgical intervention protocols include
administration of pharmaceutical agents for prophylaxis; and
surgery, including in extreme cases surgery to remove a tissue or
organ before the cancer within the organ metastasizes. Exemplary
diagnostic protocols include non-invasive and invasive imaging;
monitoring metabolic biomarkers; and biopsy screening.
[0301] For example, in some variations, the system further includes
a medical protocol database 214 operatively connected to a
computer-readable medium of the system and containing information
correlating the iASPP phosphorylation state and medical protocols
for human subjects with the cancer. Such medical protocols include
any variety of medicines, lifestyle changes, diagnostic tests,
increased frequencies of diagnostic tests, and the like that are
designed to achieve effective therapy sith minimum side effects.
The information correlating the iASPP phosphorylation state with
protocols could include, for example, information about the success
with which the metastatic cancer is avoided or delayed, if a
subject has a level of phosphorylated iASPP and follows a
protocol.
[0302] By way of example, in some variations, a system that
analyzes inputs of iASPP phosphorylation (or iASPP intracellular
localization) and at least one parameter selected from the group
consisting of: a p53 mutation that reduces a tumor suppressive
function of p53, an elevated measurement of mdm2 protein or mRNA,
and a B-RafV600E mutation, or assessing the metastatic potential of
the cancer could generate a variety of treatment protocols,
including:
[0303] for a subject with a cancer characterized by elevated iASPP
phosphorylation and/or iASPP nuclear localization, prescribing
and/or administering a therapeutic regimen that includes an
inhibitor of iASPP phosphorylation;
[0304] a therapeutic regimen that includes an inhibitor of iASPP
phosphorylation and an inhibitor of mdm2-induced degradation of
p53, for a subject with a cancer characterized by elevated iASPP
phosphorylation and/or iASPP nuclear localization and the absence
of a p53 mutation that reduces the tumor suppressive function of
p53;
[0305] a therapeutic regimen that includes an inhibitor of iASPP
phosphorylation and an inhibitor B-RafV600E, for a subject with a
cancer characterized by elevated iASPP phosphorylation and/or iASPP
nuclear localization and the presence of the B-Raf-V600E
mutation;
[0306] a therapeutic regimen that includes all three of the
foregoing agents, for a subject with a cancer that characterized by
elevated iASPP phosphorylation and/or iASPP nuclear localization,
the absence of a p53 mutation that reduces the tumor suppressive
function of p53, and the presence of the B-Raf-V600E mutation.
[0307] The system of this embodiment further includes a medical
protocol tool or routine 216, operatively connected to the medical
protocol database 214 and to the analysis tool or routine 210. The
medical protocol tool or routine 216 preferably is stored on a
computer-readable medium of the system, and adapted to be executed
on a processor of the system, to: (i) compare (or correlate) the
conclusion that is obtained from the analysis routine 210 (with
respect to metastatic potential of the cancer in the subject) and
the medical protocol database 214, and (ii) generate a protocol
report with respect to the probability that one or more medical
protocols in the medical protocol database will achieve one or more
of the goals of eliminating the cancer, slowing cancer growth or
metastasis, and minimizing deleterious side effects of cancer
treatment.
[0308] Some variations of the system just described include the
communication tool 212. In some examples, the communication tool
generates a communication that includes the protocol report in
addition to, or instead of, the conclusion with respect to
metastatic potential.
[0309] Information about the phosphorylation state of iASPP not
only can provide useful information about identifying or
quantifying potential for metastasis; it can also provide useful
information about possible causative factors for a human subject
identified with a cancer, and useful information about therapies
for the cancer patient. In some variations, systems of the
invention are useful for these purposes.
[0310] For instance, in some variations the invention is a system
for assessing or selecting a treatment protocol for a subject
diagnosed with a cancer. An exemplary system, schematically
depicted in FIG. 17, comprises:
[0311] (a) at least one processor;
[0312] (b) at least one computer-readable medium;
[0313] (c) a medical treatment database 308 operatively connected
to a computer-readable medium of the system and containing
information correlating the phosphorylation state of iASPP and
metastatic potential of the cancer;
[0314] (d) a measurement tool 306 to receive an input (304,
depicted in FIG. 17 but not part of the system per se) about the
human subject and generate information from the input 304 about the
phosphorylation state of iASPP indicative of metastatic potential
in a human subject diagnosed with the cancer; and
[0315] (e) a medical protocol routine or tool 310 operatively
coupled to the medical treatment database 308 and the measurement
tool 306, stored on a computer-readable medium of the system, and
adapted to be executed on a processor of the system, to compare the
information with respect to the iASPP phosphorylation state in a
cancer cell of the subject and the medical treatment database, and
generate a conclusion with respect to at least one of:
[0316] (i) the probability that one or more medical treatments will
be efficacious for treatment of the cancer for the patient; and
[0317] (ii) which of two or more medical treatments for the cancer
will be more efficacious for the patient.
[0318] Preferably, such a system further includes a communication
tool 312 operatively connected to the medical protocol tool or
routine 310 for communicating the conclusion to the subject 300, or
to a medical practitioner for the subject 302 (both depicted in the
schematic of FIG. 17, but not part of the system per se). An
exemplary communication tool comprises a routine stored on a
computer-readable medium of the system and adapted to be executed
on a processor of the system, to generate a communication
containing the conclusion; and transmit the communication to the
subject or the medical practitioner, or enable the subject or
medical practitioner to access the communication.
[0319] As described herein, information regarding iASPP
phosphorylation state can be advantageously combined with other
biological information about a cancer to improve evaluation of
metastatic potential, and to improve selection of treatment
regimen. Such information includes, for example, whether the cancer
contains wild type p53 or a p53 mutation that reduces a tumor
suppressive function of p53; whether the cancer expresses elevated
amounts of mdm2, and whether the cancer contains a V600E mutation
in B-Raf.
[0320] Accordingly, in some variations, the system of the invention
has a measurement tool that receives an input about the human
subject and generates information from the input about the presence
or absence of one or more parameters selected from: a p53 mutation
that reduces a tumor suppressive function of p53, an elevated
measurement of mdm2 protein or mRNA, and a B-RafV600E mutation; the
system has a database that contains population information
correlating these parameters to metastasis data and/or treatment
data; the system has an analysis tool or routine that compares the
information about the human subject relating to iASPP
phosphorylation and the one or more additional parameters; and the
system generates a conclusion with respect to metastatic potential
of the cancer and/or a treatment regimen for the cancer, using each
of the two or more types of information collected by the
system.
[0321] All publications and patents mentioned herein are hereby
incorporated by reference in their entirety as if each individual
publication or patent was specifically and individually indicated
to be incorporated by reference. In case of conflict, the present
application, including any definitions herein, will control.
[0322] The invention may be more readily understood by reference to
the following examples, which are given to illustrate the invention
and not in any way to limit its scope.
EXAMPLES
Example 1
Materials and Methods
[0323] Plasmids, Antibodies, and Cell Lines.
[0324] For protein purification pET22 plasmids encoding C-terminus
of iASPP (625-828) and ASPP2 (905-1128) with His tag were kind gift
from Ross Alexander Robinson (University of Oxford). cDNA encoding
N-terminus of iASPP (1-240 aa and 249-482 aa) with GST tag were
cloned in to pGEX between BamHI and EcoRI sites. These
constructions were expressed in bacteria and purified by nickle
beads or glutathione beads according to general protocols. Purified
cylinB1/cdk1 complex and histone H1 were kindly given by Jane
Endicott (University of Oxford). For transfected plasmids,
fragments of iASPP and ASPP2 were cloned into construction using
pcDNA3.1N5-His TOPO TA Expression Kit (Invitrogen). Primary
antibodies are from: Self-made (LX-series antibodies), Santa Cruz
(His (sc-803), p53 (sc-126), p63 (BC4A4)), Serotec (V5 (MCA1360)),
and Cell signaling (cleaved PARP (9541), total PARP(9532), cleaved
Caspase-3 (9661S), total Caspase-3 (9662)).
[0325] Most of cell lines were available in our lab store. IGR37
and IGR39 were kindly given by prof. Colin (University of Oxford).
Prostate cancer cell lines were collected by Dr Richard (University
of Oxford). iASPP knock out MEFs were prepared from embryonic day
13.5 embryos derived from iASPP knock out mice (Notari et al.,
2011), and immortalized by Ras/E1A transforming.
[0326] Western Blotting (WB) and Immunoprecipitation:
[0327] For WB, cells were lysed in buffer containing 8 M urea, 1 M
thiourea, 0.5% CHAPS, 50 mM DTT, and 24 mM spermine. A total of 40
.mu.g of protein extract was loaded per lane into
SDS-polyacrylamide gels. Gels were transferred onto nitrocellulose
membrane (Protran) and the resulting blots incubated first with
primary antibody overnight at 4.degree. C. and then with the
appropriate secondary horseradish peroxidize-conjugated antibody
(Dako). The results were visualized by enhanced chemiluminescent
detection (Amersham Biosciences) using X-ray film (Fujifilm). For
cell lysate immunoprecipitation, cells were dissolved in NP40
buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% NP-40) with protease
inhibitor cocktail mix (Roche Diagnostics). Cells were further
thoroughly broken by ultrasonication, followed by spinning for 1
hour. Achieved suspension was adjusted to 1-2 mg/ml using NP40
buffer and incubated with 20 .mu.l nickel beads, glutathione beads,
or protein A beads (with 2 .mu.g required antibody) for 2 h at room
temperature. The beads were washed for four times with NP40 buffer,
boiled for 5 min in Laemmli sample buffer, and applied for Western
blotting. For pure protein immunoprecipitation, 5-10 .mu.g purified
proteins were mixed in NP40 buffer with 1 mg/ml BSA for 2 h at room
temperature.
[0328] p53 Sequencing:
[0329] Total mRNA was extracted from melanoma cell pellets using
Qiagen RNeasy Mini-Kit (Cat No 74104). cDNA was then amplified
according to protocol for Invitrogen SuperScriptIII Reverse
Transcriptase (Cat No 18080). Full length p53 were amplified from 5
.mu.l cDNA using primers p53-1F (5'-CGGGGACACTTTGCGTTC-3' SEQ ID
NO: 3) and p53-1R (5'-TTCTGACGCACACCTATTGC-3' SEQ ID NO: 4). The
PCR product was sequenced using primers p53-2F
(5'-GCTTTCCACGACGGTGAG-3' SEQ ID NO: 5) and p53-2R
(5'-CAAGGGTTCAAAGACCCAAA-3' SEQ ID NO: 6).
[0330] In Vitro Cyclin B1/Cdk1 Assay:
[0331] 5 .mu.g iASPP fragment was incubated with 100 ng cyclin
B1/cdk1 complex and 1 .mu.Ci [.gamma.-32P]-ATP in kinase assay
buffer for 10 min at room temperature. Reaction was stopped by SDS
running buffer and subjected for SDS-PAGE gel. After
electrophoresis, gels were fixed, dried and subjected to
autoradiography.
[0332] FITC Protein Labeling and In Vitro Nuclear Import Assay:
[0333] Purified proteins were labeled with FITC according to
protocol from protein label kit (F-6434, Invitrogen).
Permeabilization and in vitro import assay were mainly carried out
as described (Van Impe et al., 2008): digitonin treated
semi-permeable cells (pre-seed on coverslides) were incubated in
NIB buffer with 0.02 mg/ml FITC labelled substrate and 10 mg/ml
H1299 cytoplasm factors at RT for 40 min. The cellular localization
of the substrate was determined by direct fluorescence observation
under confocol microscope. NIB buffer: (20 mM HEPES, pH 7.3, 110 mM
potassium acetate, 5 mM sodium acetate, 2 mM magnesium acetate, 2
mM DTT, 0.5 mM EGTA and protease inhibitors). For iASPP(1-240)
pretreated experiment, 0.02 mg/ml FITC labeled substrates were
incubated with 0-1 mg/ml iASPP(1-240) at room temperature for 2
hours, followed by nuclear import assay.
[0334] Tissue Section Staining and Cell Immunofluorescence
Staining:
[0335] As described in Notari et al., 2011, the disclosure of which
is incorporated by reference in its entirety. Briefly, Tissue
sections were dewaxed, rehydrated and incubated with 3% hydrogen
peroxide in methanol to block endogenous peroxidase activity (10
mins). Prior to this incubation, antigen retrieval was carried out
in microwaved sodium citrate buffer (pH 6.0) for 10 mins for cyclin
B1 staining only. Sections were then blocked with normal goat serum
(NGS), incubated overnight at 4.degree. C. with the primary
antibody, followed by the biotinylated secondary antibody for 40
minutes at room temperature. The Avidin-Biotin
immunoperoxidase-Complex Vectastain Elite Reagent was used to
amplify the signal and the peroxidase substrate Vector VIP was used
to visualize the complexes. The sections were then counterstained
with Methyl Green, dehydrated, permanently mounted and
photographed. The expression of iASPP in the nucleus was scored as
the product of staining intensity (0--none, 1--weak, 2--moderate,
3--strong) and the proportion of cells exhibiting this stain
(0-100% in 5-10% increments) used to generate an Overall Expression
score (0-300). All tumor cells showed cyclin B1 expression so
expression levels were compared using an intensity score only
(1-weak, 2-moderate, 3-strong).
[0336] Statistical analysis was undertaken in SPSS. Differences
between groups were analysed by the Mann-Whitney test (U) where the
effect size is denoted by r=z/ n(>0.3=medium effect,
>0.5=large effect). The relationship between nuclear iASPP,
dichotomized around the upper quartile, in primary and metastatic
tumors was evaluated using a Chi-squared test where the effect size
is determined by the odds ratio. For survival analysis, a
multivariate Cox proportional hazards model was used, controlling
for known prognostic factors (age, gender, site, thickness,
ulceration).
[0337] Mass Spectrometry Assay.
[0338] Isolated gel bands were subjected to trypsin digestion as
described previously (Batycka et al., 2006). For the analysis of
digested protein material, liquid chromatography was performed
using an Ultimate 3000 nano-HPLC system (Dionex). Samples were
concentrated on a trapping column (LC Packings, 300 .mu.m ID, 0.1
cm) at a flow rate of 25 .mu.l/min. For the separation with a C18
Pepmap column (75 .mu.m ID, 15 cm, LC Packings), a flow rate of 300
nl/min was used as generated by a cap-flow splitter cartridge
(1/300). Peptides were eluted by the application of a 30 min linear
gradient: solvent A (95% H2O, 5% acetonitrile, 0.1% formic acid),
0-45% solvent B (95% acetonitrile, 5% water, 0.1% formic acid). LC
was interfaced directly with a 3D high capacity ion trap mass
spectrometer (HCTultra, Bruker Daltonics) utilizing 10 .mu.m ID
distal coated SilicaTips (New Objective) and nano-ESI mode. MS/MS
analysis was initiated on a contact closure signal triggered by
HyStar.TM. software. Up to 4 precursor ions were selected per cycle
with active exclusion (0.5 min.). Raw LC-MS/MS data were processed
and Mascot compatible files created using DataAnalysis 3.4 software
(Bruker Daltonics). The MS/MS data representing phophorylated
peptides was inspected manually and was performed according to
published guidelines (Kamath et al., 2011).
[0339] Generation and Epitope Mapping of Mouse Monoclonal
Anti-iASPP Antibody.
[0340] The immunogen used to generate iASPP mouse monoclonal
antibodies LX49.3 and LX128.1-10 were human iASPP (439-639)-His
fragment and human iASPP (1-240)-GST fragment, respectively. To map
their epitopes, iASPP peptides were coated in 96-well plate and
probed with above antibodies. The peptides recognized by LX 49.3,
LX128.1, LX128.5, LX128.6, LX128.7, LX128.8, and LX128.9 were as
follows: iASPP (501-510), iASPP (111-120), iASPP (111-122), iASPP
(36-45), iASPP (36-45), iASPP (96-105), and iASPP (96-105),
respectively.
[0341] Generation of and Assays Determining Activity of iASPP-Ser
113 Phospho-Specific Antibodies:
[0342] iASPP-Ser 113 phospho-specific antibodies were generated by
immunizing rabbits with an iASPP-Ser-113 phospho-specific peptide
(NH.sub.2--HPYSPL[phospho-S]PKGRPSC-CONH.sub.2 (SEQ ID NO: 14,
which corresponds to amino acids 107-118 of SEQ ID NO: 2 (FIG. 23A)
with a C-terminal cysteine added to allow conjugation to KLH). The
sequence selected for immunization is conserved between human and
mouse iASPP. Generated antibodies were individually purified
against both phospho-modified and non-modified peptide.
[0343] The phosphor-specific ser 113 anti-iASPP antibody (from
rabbit 42310) was tested for binding activity by evaluating
nocodazole (1 .mu.M)-induced cell cycle arrested human U2OS cells
(FIG. 23B). Human U2OS cells were treated with nocodazole (1 .mu.M)
at the time points indicated (FIG. 23B), all cells in the culture
dish were harvested and lysate prepared by incubating for 30
minutes at room temperature in 8M urea, 1M thiourea, 0.5% CHAPS, 50
mM DTT and 24 mM spermine, followed by centrifugation at 20 000 g
for 20 min. For the floating and adherent cells, the rounded
(mitotic rich) cells were separated from the adherent cells after
24 hours treatment with nocodazole. Lysates were separated on a 6%
SDS PAGE gel and proteins analysed by conventional western
blotting. iASPP phosphor-ser113 was detected using the antibody
from rabbit 42310 as described in FIG. 23A, and total iASPP was
detected by re-probing the blot with antibody LX049.3. Nucleolin
was used as a loading control. Binding of phosphor-specific ser 113
anti-iASPP antibody (42310) was demonstrated by evaluating the
accumulation of phosphorylated iASPP over time in nocodazole
arrested mitotic cells (FIG. 23B). Consistent with enhanced iASPP
phosphorylation in nocodazole arrested mitotic cells, enhanced
iASPP phosphorulation on ser 113 was also observed in the floating
cells, which are mostly mitotic cells, and not in adherent cells
(FIG. 23B). Total iASPP was detected by re-probing the blot with
antibody LX049.3.
[0344] Additionally, the specific binding of iASPP antibody (42310)
to phosphorylated ser 113 were evaluated by determining the failure
of iASPP antibody (42310) to bind when the protein is not
phosphorylated at ser 113. Since CDK kinase phosphorylates iASPP at
ser 113, CDK inhibitor JNJ-7706621 was used to treat human U2OS
cells. Binding was compared between untreated or JNJ-7706621
treated cells that were incubated for 16 hours with nocodazole (1
.mu.M). Western blotting was carried out as described in FIG. 23B
using an 8% SDS PAGE gel. When compared to untreated cells, binding
of phosphor-ser 113 specific anti-iASPP antibody was not observed
in the presence 0.5 .mu.M JNJ-7706621 (FIG. 23C). Total iASPP was
detected by re-probing the blot with antibody LX049.3. This
experiment demonstrated the requirement for phosphorylation on
serine 113 on iASPP in order for the antibody to bind. The western
blot showed a decreased in binding of phosphor-specific ser 113
anti-iASPP antibody (42310) in the presence of CDK inhibitor
JNJ-7706621 (FIG. 23C).
[0345] Generation of iASPP-Ser 84 Phospho-Specific Antibodies:
[0346] iASPP-Ser 84 phospho-specific antibodies will be generated
by immunizing rabbits with an iASPP-Ser 84 phospho-specific peptide
(NH.sub.2--PEPFGSRG[phospho-S]PRKAATC-CONH.sub.2, (SEQ ID NO 15,
which corresponds to amino acids 76-90 of SEQ ID NO: 2 with a
C-terminal cysteine added to allow conjugation to KLH). Generated
antibodies will be purified against both phospho-modified and
non-modified peptide.
[0347] p53 and iASPP Knock Down.
[0348] P53 siRNA (MU-003329-03-0002, Dharmacon), or iASPP siRNA
(MU-003815-01-0002) was wet reverse transfected into cells using
DharmaFECT 1 Transfection Reagent kit (T-2001-02) during cell
seeding. Protein levels were determined by WB.
[0349] Cell Cycle Analysis by Flow Cytometry.
[0350] Cells were harvested, washed and fixed in ice-cold 70% EtOH.
Cells were washed and resuspended in PBS containing 0.05 mg/ml
RNase A and 0.05 mg/ml propidium iodide (P4864, Sigma). After 30
min RT incubation cells were analyzed by flow cytometry.
[0351] Colony Assay.
[0352] After drugs treatment, cells were digested, counted, and
seed in 6-well plates (100, 1 000, 10 000 cells/well). Cells were
then grown for 10-14 days and stained by Giemsa (GS-500,
Sigma).
Example 2
P53 Selectively Binds Slow-Migrating Nuclear iASPP
[0353] An inverse correlation between nuclear iASPP and epithelium
differentiation in vivo illustrates the importance of iASPP
localization in regulating its function (Notari et al., 2011). To
understand the regulation of iASPP functions in vivo, it is
critical to identify experimental systems in which iASPP
localization is regulated. To achieve this, a panel of human tumor
cell lines was examined for their iASPP expression patterns. In
osteosarcoma, epithelium, breast, and renal cancer cell lines,
iASPP is predominantly expressed as cytoplasmic protein (FIG. 2).
Interestingly, however, a large number of melanoma cell lines
express nuclear iASPP (FIGS. 1A and 1B). While SK-MEL37, WM278, and
IGR39 show more cytoplasmic staining, SK-MEL23, WM115, and IGR37
(metastatic cell line derived from the same patient as that derived
for IGR39) show predominantly nuclear staining. iASPP nuclear
staining pattern is also found in LNCaP3, a cell lines derived from
LNCaP after 3 rounds of ex-vivo selection of metastasis (Pettaway
et al., 1996). To identify the underlying reason for the observed
difference in iASPP cellular localization, iASPP proteins were
analyzed by immunoblotting. Interestingly iASPP migrated as two
bands in many melanoma cell lines tested and a slow-migrating iASPP
isoform was tightly associated with the appearance of nuclear iASPP
(FIG. 1B). While SK-MEL37, WM278, and IGR39 express more fast
migrating iASPP isoform (black arrows), SK-MEL23, WM115, and IGR39
express predominantly slow-migrating iASPP (red arrowheads), so
does LNCaP3. This is in contrast to the single iASPP band (fast
migrating band) detected in all other cell panels that
predominantly express cytoplasmic iASPP (FIG. 2, Table 1 of FIG. 2B
and other data not shown).
[0354] Knowing that iASPP is able to bind and inhibit the apoptotic
function of p53 and that p53 is predominantly a nuclear protein,
the binding preference of p53 to different iASPP isoforms was
evaluated. p53 was immunoprecipitated in a panel of melanoma cells
that expressed various amount of nuclear iASPP with different
migration ability. The results show that p53 was able to
selectively co-immunoprecipitate the slow-migrating iASPP isoform
in these melanoma cells (FIG. 1C). Under the same conditions p53
failed to co-immunoprecipitate the fast migrating iASPP. The
failure of p53 to co-immunoprecipitate fast migrating iASPP is not
linked to the level of iASPP isoform nor to the amount of
precipitated p53 (FIG. 1C). These data suggest that p53
preferentially binds slow-migrating iASPP that is localized in the
nucleus.
Example 3
Mitotic Arrests Induces Slow-Migrating Nuclear iASPP
[0355] To better understand the signals that induce slow-migrating
nuclear iASPP, a p53-null cell line H1299 was first used to study
iASPP expression in response to various chemotherapeutic agents
Like the majority of tumor cell lines studied, H1299 predominantly
expresses cytoplasmic iASPP that migrates as a single band (FIG.
3A). Interestingly, the slow-migrating iASPP was induced only on
treatment with mitotic arresting reagents such as Nocodazole, Taxol
and Colchicine. In contrast, treatment with other chemotherapeutic
agents that do not induce mitosis, such as Doxorubicin,
Staurosporine, Cisplatin and hydrogen peroxide, did not result in
increased slow-migrating iASPP isoform expression. As mitotic cells
often detach from the tissue culture dish, floating cells were
separated from adherent cells after the treatment of Nocodozole,
Taxol and Colchicine. FIG. 3B shows the enrichment of
slow-migrating iASPP in the floating mitotic cell population
whereas iASPP is expressed as a single band in the adherent cells
even after drug treatment.
[0356] To provide further evidence that slow-migrating iASPP was
induced by mitosis and is not limited to H1299 cells, mitotic H1299
and MCF7 cells were generated by mitotic shake, and enrichment of
G2/M phase was confirmed by FACS analysis. Again, slower migrating
iASPP was only detected in the collected mitotic cells, whereas in
adherent cells iASPP is predominantly expressed as a single band
(FIG. 3C). Importantly when the cellular localization of iASPP was
examined in these two cell populations, it was observed that
mitotic cells predominantly express nuclear iASPP whereas adherent
cells predominantly express cytoplasmic iASPP (FIG. 3C).
[0357] Finally, to confirm that the slow-migrating iASPP observed
in melanoma cell lines was induced by the same signals, a panel of
melanoma cell lines that expressed both slow and fast migrating
iASPP isoforms were treated with Nocodazole to induce mitosis.
Again, Nocodazole induced the conversion of faster migrating iASPP
into the slower migrating iASPP isoform (FIG. 3D), suggesting that
the slow-migrating nuclear iASPP is the same as that induced by
mitosis. To provide further evidence that slow-migrating iASPP is
specifically induced by mitotic arrest H1299 cells were generated
by mitotic shake, and enrichment of G2/M phase was confirmed by
FACS analysis (FIG. 4A). To eliminate the possibility that slow
migrating iASPP is induced by cell detachment, H1299 cells were
treated with stimuli such as EDTA or cold shock that induce cell
detachment but not mitosis. Consistent with our findings above,
both EDTA and cold shock failed to affect iASPP mobility, even
though under the same conditions nocodazole induced slow migrating
iASPP (FIG. 4B).
Example 4
Phosphorylation of iASPP at Ser84/Ser113 by Cyclin B1/Cdk1
Contributes to iASPP Slow Migration
[0358] To identify the regions of iASPP that are responsible for
mitosis induced slow migration, two V5 tagged iASPP truncation
mutants; iASPP (1-478)-V5 and iASPP (482-828)-V5 were transfected
into H1299 cells. The transfected cells were treated with
Nocodazole or Taxol to induce mitosis. The mobility shifts of the
transfected iASPP mutants were detected using anti-V5 antibody. The
results shown in FIG. 3A illustrate that slow-migrating
iASPP(1-478)-V5 was specifically induced in Nocodazole or Taxol
treated cells. However, under the same conditions, iASPP
(482-828)-V5 appeared predominantly as a single band, indicating
that modifications on the N-terminal half of iASPP are responsible
for the mitosis-induced slow migration of iASPP.
[0359] Phosphorylation is one of the best known modifications that
can induce protein mobility shifts, and cyclin B1/cdk1 is one of
the most active kinases in mitosis. We therefore tested whether
iASPP is a substrate of cyclinB1/cdk1 and whether this
phosphorylation may affect iASPP migration. Recombinant N-terminal
iASPP fragments; iASPP (1-240) and iASPP (249-482) were tested in
an in vitro kinase assay using purified cyclin B1/cdk1. iASPP
(1-240) but not iASPP (249-482) bound and was phosphorylated by
cyclinB1/cdk1 in vitro (FIG. 5B). The amino acid sequence that
covers the N-terminal of amino acids 1-240 of iASPP was analyzed by
Motifscan and identified S84, S113, and S120 of iASPP as potential
cyclinB1/cdk1 phosphorylation sites. Thus Ser84, Ser113 and Ser120
were singly or doubly mutated to Alanine. The resulting iASPP
(1-240) mutants (S84A, S113A; S120A; S84A/S113A; S113A/120A) were
used in the cyclinB1/cdk1 kinase assay in vitro. Results shows that
cyclinB1/cdk1 failed to phosphorylate iASPP (1-240) S84A/S113A
whereas all other phosphorylation mutants of iASPP can be
phosphorylated by cyclinB1/cdk1 in vitro.
[0360] The two iASPP phosphorylation mutants; iASPP(S84A/113A)-V5
and iASPP(S84D/113D)-V5 were also transfected into melanoma cell
lines IGR37 and IGR39. As expected, slow-migrating iASPP-V5 (wild
type) was detected in IGR37 (without treatment of Nocodazole), but
not in IGR39 cells. Interestingly, iASPP(S84D/113D) produced some
slow-migrating iASPP in IGR37 cells, but not in IGR39 cells. In
both iGR37 and IGR39 cells, iASPP(S84A/113A) remained as a single
band (FIG. 5D).
[0361] Finally, mass spectrometry analysis confirmed that
endogenous iASPP from H1299 cells was phosphorylated at 5113, but
not during interphase (FIG. 6). Taken together, these findings
demonstrate that iASPP is phosphorylated by cyclinB1/cdk1 at Ser84
and Ser113 and that phosphorylation retards iASPP migration.
[0362] Discussion:
[0363] CyclinB1/cdk is involved in iASPP modification during
mitosis and in melanomas. According to whole genome based arrays
examining the genes aberrantly expressing in melanoma cell lines in
vitro, cyclin B1 was of the most upregulated genes (fold change:
+18.17) (Avery-Kiejda et al., 2011). In vivo, cyclin B1 had
dramatically increased protein expression in metastases tissue
sections comparing with primary melanomas (Georgieva et al., 2001).
In esophageal squamous cell carcinoma, cyclin B1 has been
implicated in metastasis by promoting an epithelial-mesenchymal
transition (Song et al., 2008). It seems these overexpressed cyclin
B1 play some other roles (such as dominate phosphorylation pattern
over unphosphorylation pattern for its substrates) besides
functioning as mitosis regulator as the melanoma cells universally
show normal cell cycle profile. Supporting the proposed role of
cyclin B1/cdk1 in vitro, levels of modified iASPP showed
significant positive correlation with cyclinB1 level (activator of
cdk1 kinase), but not cdk1 level, in 18 melanomas, 7 prostate
cancer cells, and in mitotic H1299 cells (FIGS. 10 and 11).
Supporting cdk1 's role in iASPP phosphorylation, phosphorylation
kinase inhibitor screening result shows that cdk and clk (cdk like
kinase) inhibitors can indeed prevent iASPP modification, while
other kinase inhibitors (Aurora, Plk1, or Pim 1) cannot (FIG. 12).
Herein, a cdk1 inhibitor, k00616a was highlighted as its high
efficiency in blocking iASPP modification. It has been reported to
be able to inhibit melanomas in vivo in a human tumour xenograft
model in mice (Emanuel et al., 2005).
[0364] Besides cyclinB1 and cdk1, the expression pattern of other
24 key proteins involved in tumorgenesis of melanomas were
screened. Among these 24 proteins, the interested ASPP2, MDM2
family members, melanoma-associated antigens, familial melanomas
candidate p16ink4a, Braf-Brn2-MITF-TBX2 pathway components, mitosis
related cdks and cyclins, ckd5 and regulators, and prolyl
hydroxylases did not show significant correlation with iASPP
modification pattern, except for p53 and MAGE-C2. While MAGE-C2
showed relatively weak correlation with iASPP modification pattern,
p53 showed a strong correlation with iASPP modification pattern in
both 18 melanomas and 7 prostate cancer cells. p53 cDNA were then
amplified from melanomas and their statuses were sequenced. As a
result (FIG. 10C and FIG. 11C), cells with a low ratio of modified
iASPP universally host mutated p53 (6/7 for melanomas, 5/5 for
prostate. Me248.3 is the only exception; it hosts wild type p53
mRNA, but exhibits null p53 protein level for an unknown reason.
Cells with high ratio of modified iASPP universally host wt p53
(10/11 for melanomas, 2/2 for prostate). IGR37, the only exception
with mutated p53 but a high iASPP modification ratio, is derived
from a same patient with primary IGR39. IGR37 is established to be
a malignant melanoma cell line, described to be very metastatic and
tumorigenic in nude model and confirmed in human. It shows high
iASPP modify ratio comparing with primary IGR39 (.about.80% vs
.about.15%). It implied that iASPP might also be preferentially
modified in metastatic cells. This implication was confirmed in
prostate LNCaP3, the cell line derived from LNCaP after 3 times
metastasis in mouse. Consistent with IGR37/39 couple, LNCaP3 show
significantly increased iASPP modify ratio comparing with LNCaP
(FIG. 10C, .about.75% vs .about.35%, FIG. 11C). The trend was also
partially fit in head and neck cell couples, though it is not as
dramatic as in melanomas or prostate (data not shown).
Example 5
S84/S113 Phosphorylation of iASPP Promotes iASPP Nuclear
Localization by Preventing N- and C-Terminal Self-Interaction
[0365] In our recently solved crystal structure of C-terminus of
iASPP, we observed that a peptide (GSPRKARR) within iASPP (residues
615-622 of SEQ ID NO: 2) was able to efficiently bind C-terminal
iASPP and mask the p53 binding space (Robinson et al., 2008).
Interestingly, this peptide shares high sequence similarity with
the N-terminal region of iASPP where S84 (GSPRKAAT, amino acids
83-90 of SEQ ID NO: 2) is located. This led us to the hypothesis
that the N-terminal of iASPP interacts with its C-terminus, and
phosphorylation of iASPP at Ser84 and Ser113 may interfere with
this interaction. Indeed, purified iASPP(1-240) but not
iASPP(249-482) can interact with purified iASPP C-terminal
fragment, iASPP(626-828), which contains the ankryin repeats and
SH3 domain. Under the same conditions, iASPP(1-240) did not
interact with the C-terminus of ASPP2 (905-1128, containing ARDs
and SH3 domain) (FIG. 7A). Using a number of iASPP variants singly
and doubly mutated at Ser84, Ser113 and Ser120 sites it was
observed that residues S84 and S113, but not S120, are involved in
this N- and C-terminal interaction of iASPP as Ala mutation of
Ser84 and Ser113 largely weaken the interaction (FIG. 7A, lower
panel: compare lanes 2 versus 5 and 6). Phosphorylation mimic
mutations Ser84D and Ser113D efficiently abrogated this interaction
(FIG. 7A, lower panel: compare lanes 2 versus 9).
[0366] The involvement of S84/S113 phosphorylation in iASPP N- and
C-term self-interaction in vivo was further tested by transfecting
N-terminal iASPP(1-478) into Ras and E1A immortalized iASPP (-/-)
mouse embryonic fibroblasts (MEFs) and the transfected cells were
treated with nocodazole to induce post-translational modification
and slow migration of the transfected iASPP(1-478). The transfected
and treated cell lysates were incubated with the purified
recombinant iASPP C-terminal fragment, iASPP(625-828). Consistent
with the prediction that phosphorylation at Ser84/Ser113 disrupts
the N- and C-terminal self-interaction of iASPP, the iASPP(625-828)
selectively bound the non-modified, fast migrating N-terminal iASPP
(FIG. 7B). Similarly, when purified recombinant iASPP(1-240) was
incubated with nocodazole treated H1299 cell lysates, it
specifically bound to nocodazole-induced modified, slow-migrating
endogenous iASPP (with exposed C-terminus, FIG. 7B).
[0367] To understand whether iASPP forms N- and C-terminal
self-interacting monomers or anti-parallel homodimers, iASPP
migration was compared between non-denaturing or denaturing gels
using H1299 cell lysates derived from nocodozale treated or
non-treated cells. In a denaturing SDS-page gel, iASPP migrated as
a single 100 kd band in normal growing H1299, but migrates as
double bands in nocodazole treated H1299 cells. In a non-denaturing
gel, iASPP from normal growing H1299 cells migrated as a single
band but with a molecular weight almost double the size detected in
SDS denaturing gel (FIG. 7B). The iASPP expressed in Nocodazole
treated H1299 cells migrated as two bands that differed
dramatically in size, one was about 90 kd whereas the other
migrated at about 180 kd, almost double the size of the fast
migrating iASPP (FIG. 7B). This result suggests that in normal
growing H1299 cells, iASPP exists as an anti-parallel homodimer.
However, in mitosis and upon Ser84/Ser113 phosphorylation, the N-
and C-termini self interaction is disrupted and results in the
formation of an iASPP monomer.
[0368] Based on the crystal structure of iASPP and the sequence
similarity between two iASPP peptides, (GSPRKAAT, amino acids 83-90
of SEQ ID NO: 2) and (GSPRKARR, amino acids 615-622 of SEQ ID NO:
2), it was suggested that T722, L724, N813, and Y814 might be
contact residues located at the C-terminal end of iASPP. Single and
double mutants were generated on these four residues and their
ability to interact with N-terminal iASPP(1-240) was tested in
vitro. We observed that iASPP(625-828)/T722A and
iASPP(625-828)/L724A can bind N-terminal iASPP slightly less
efficiently than wild type iASPP(625-828). However
iASPP(625-828)/N813A or iASPP(625-828)/Y814A have a much reduced
ability to bind iASPP(1-240). Additionally,
iASPP(625-828)/T722A/N813A, iASPP(625-828)/T722A/Y814A,
iASPP(625-828)/T724A/N813A or iASPP(625-828)/T724A/Y814A almost
lost their ability to interact with iASPP(1-240). This identifies
N813 and Y814 as two key residues that interact with N-terminal
iASPP (FIG. 7C, and FIG. 8).
[0369] To test the hypothesis that N- and C-terminal
self-interaction prevents iASPP nuclear entry, digitonin
permeabilized H1299 cells (with permeable cellular membrane and
intact nuclear membrane) were used to carry out an in vitro nuclear
entry assay using FITC labeled iASPP C-terminal fragment;
iASPP(625-828). FITC labeled ASPP2 C-terminus; ASPP2(905-1128) was
used as a negative control. As reported previously (Slee et al.,
2004), both C-terminal iASPP and ASPP2 can enter the nucleus with
high efficiency. Interestingly, a pre-incubation with increasing
amount of non-labeled N-terminal iASPP(1-240) effectively prevented
the nuclear entry of FITC labeled iASPP(625-828). Under the same
conditions, a pre-incubation of iASPP phosphor-mimic mutant,
iASPP(1-240)S84D/S113D, failed to have any impact on the nuclear
entry efficiency of FITC labeled iASPP(625-828). Furthermore,
iASPP(1-240) only affected the nuclear entry of iASPP(625-828)
since it had no impact on the nuclear entry of ASPP2(905-1128)(FIG.
7D). In agreement with this, iASPP(S84D/S113D), showed more nuclear
localization comparing with wild type iASPP in iASPP(-/-) MEFs
(FIG. 7E).
Example 6
Phosphor-Mimic iASPP, iASPP(S84D/S113D), is More Active than Wild
Type iASPP in Binding and Inhibiting p53
[0370] Knowing that modified iASPP unmasks its nuclear localization
and p53 binding sites, it was hypothesized that phosphor mimic
iASPP double mutant, iASPP(S84D/S113D), would bind p53 more
effectively both in vitro and in vivo. This binding affinity
between iASPP wild type and iASPP(S84D/S113D) was first compared
using in vitro translated iASPP and recombinant p53. The results
shown in FIG. 9A illustrate that iASPP(S84D/S113D) was able to bind
p53 much better than iASPP wild type. When transfected into H1299
cells, the phosphor-mimic mutant, iASPP (S84D/S113D), was also able
to bind co-transfected p53 much more effectively (FIG. 9B). In
agreement with these findings, iASPP(S84D/S113D), was observed to
be .about.3-fold more effective in inhibiting p53 mediated
transcription on promoters such as PIG3 (FIG. 9C).
[0371] The anti-apoptotic function of iASPP and its mutant
iASPP(S84D/S113D) was tested in Ras and E1A transformed
iASPP(-/-)MEFs (referred to as iASPP(-/-)MEFs). Cells that stably
expressed either vector, iASPP or iASPP(S84D/S113D) were treated
with DMSO, Nutlin3 or 10 J/m2 of UV light. Apoptosis was measured
by the presence of cleaved PARP (c-PARP) or active caspase 3
(c-Caspase 3). The expression levels of iASPP and its mutant are
similar and comparable to that of endogenous iASPP (data not
shown). The presence of iASPP or iASPP(S84D/S113D) had little
impact on cell survival in DMSO treated cells. However, when the
panel of cells were treated with Nutlin3 to induce endogenous p53,
apoptosis was detected in vector transfected iASPP(-/-)MEFs, based
on an increase in c-PARP and c-Caspase 3 and a decrease in pro-PARP
and pro-caspase (FIG. 9D, compare lane 1 versus lane 4). The
presence of iASPP caused a small decrease in c-PARP expression but
had minimal impact on c-caspase 3 expression (compare lanes 4 and
5). The presence of iASPP(S84D/S113D), however, decreased the
expression levels of both c-PARP and c-caspase 3 (FIG. 9D, compare
lanes 4 and 6). A decrease in c-caspase 3 was also observed for
Cisplatin or Etopside treatment (FIG. 9E). These results suggest
that iASPP(S84D/S113D) is more potent than wild type iASPP in
inhibiting p53 induced apoptosis in response to Nutlin3, Cisplatin,
or Etopside treatment. Similarly, the presence of iASPP(S84D/S113D)
also resulted in the highest reduction in the levels of c-PARP and
c-caspase 3 in comparison to that detected in vector or iASPP
expressing cells exposed to 10 J/m2 UV (FIG. 9D, compare lanes
7-9).
Example 7
Ser84/Ser113 Phosphorylated iASPP is Enriched in Wild Type p53
Expressing Tumor Cells with High Metastatic Potential
[0372] Based on the findings presented above, it was hypothesized
that nuclear iASPP is associated with cancer malignancy. This
hypothesis was tested in a human melanoma tissue microarray (FIG.
10A). It was observed that iASPP is predominantly distributed in
the nucleus in most melanoma metastases, while it is relatively
equally distributed between the nucleus and the cytoplasm in
primary melanomas. Further, the overall nuclear iASPP level is
significantly higher in lymph node metastases (Mdn70.0) compared to
primary tumours (Mdn40.0) (U=1740.000, r=-0.21, p<0.01).
[0373] To confirm that the observed alterations in iASPP location
and function are at least partly caused by modifications on the
identified region of iASPP that contains Ser84 and Ser113, we
generated a panel of mouse monoclonal anti-iASPP antibodies with
epitopes mapped within the identified modification region of iASPP
(Residues 81-130 of SEQ Id NO: 2, FIG. 11A). Only fast-migrating
cytoplasmic iASPP was recognized by LX128.5 antibody (FIG. 10B).
LX49.3 recognizes iASPP outside the modified region whereas LX128.5
has an epitope that locates within the modified region (i.e., amino
acids 81-130 of SEQ ID NO: 2) and modification of iASPP on S84 and
5113 destroy its epitope. Thus, LX49.3 and LX128.5 indirectly
allowed us to study the phosphorylation status of iASPP in vivo.
Using these mouse monoclonal antibodies it was confirmed that both
nuclear iASPP and slow-migrating iASPP (also iASPPS84D/S113D) lost
the epitope of LX128.5, which was recognized by LX49.3 previously
(FIG. 11A). By comparing to the expression patterns of iASPP in the
same panel of 18 melanoma cell lines we were able to conclude that
the slow migrating iASPP detected by LX49.3 all lost their LX128.5
epitope. Thus, the slow migrating iASPP detected in this panel of
cell lines are likely to be phosphorylated nuclear iASPP that is
modified in the region that contains Ser84 and Ser113 (FIG. 11A,
lower panels).
[0374] Knowing that phosphorylated nuclear iASPP is more potent in
binding to p53 and inhibiting its function, we tested whether
cyclinB/cdk1 phosphorylated nuclear iASPP contributes to the
inactivation of p53 in highly metastatic melanomas. The panel of 18
melanoma cell lines used above were analyzed for iASPP, p53, and
cyclin B1 expression. Strikingly, 91% (10/11) of the wild type
p53-expressing melanoma cell lines also expressed a high ratio of
phosphorylated iASPP (>50%). In contrast, 86% (6/7) of the
mutated p53-expressing melanoma cell lines expressed low ratios of
phosphorylated iASPP (<20%, FIG. 6A). Additionally, around 80%
(9/11) of the wild type p53 cells also expressed detectable to high
levels of mdm2. However, only around 45% of wild type p53 melanoma
cell lines expressed detectable mdmX (5/11) under the same
conditions. Importantly, around 70% of the wild type p53 melanoma
cell lines express relatively high levels of mdm2 and
phosphorylated nuclear iASPP. Many of the wild type p53 cell lines
that expressed high levels of phosphorylated iASPP also expressed
relatively high levels of cyclin B1 (FIG. 11B, bar graph). In
contrast to the cyclin B1 level, the levels of phosphorylated iASPP
were not linked to the expression levels of other cell cycle
regulating proteins such as cdk1, cdk2, cyclin A, cyclin E, p16,
p21 and p27 (FIG. 11B). The elevated cyclin B1 expression in a
large number of cell lines is not simply due to an increase in the
number of cells in mitosis, as the expression levels of
phospho-histone H3 were different from those of cyclin B1 (FIG.
11B, lower panel), indicating that some of the melanoma cell lines
have deregulated cyclin B1. Together the results suggest that
deregulated cyclin B1/cdk1 may phosphorylate iASPP in interphase
and enhance its ability to inhibit p53. Thus, p53 in melanoma cells
is likely to be inactivated by both mdm2 and phosphorylated nuclear
iASPP.
[0375] Using a number of paired human tumor cell lines derived from
primary and metastatic tumors from the same patients, it was
observed that modified iASPP is enriched in metastatic tumor cells
(FIG. 10D). High levels of modified iASPP were also observed in a
panel of prostate cancer cell lines PC3, DU145, LNCaP, LNCaP3,
DuCaP, VCaP, RWPE1 (FIG. 11C). There was also a close association
between modified iASPP level and cyclin B1 level in panels of 7
prostate cancer cell lines (FIG. 11C).
Example 8
Restoring the Tumor Suppressive Function of p53 and Inhibiting
BRAF-V600E Causes Growth Suppression of Melanoma Cells
[0376] As the majority of melanoma cell lines expressing wild type
p53 also express phosphorylated nuclear iASPP, high levels of mdm2
and cyclin B1/cdk1, we hypothesised that cyclin B1/cdk1 inhibition
together with Nutlin3 could reactivate p53 function by preventing
iASPP and mdm2 from inhibiting p53 in melanoma cells. To achieve
this, we needed to identify inhibitors that could prevent iASPP
phosphorylation. A panel of 18 G2/M kinase inhibitors were screened
for their ability prevent iASPP modification in H1299 cells, in the
presence of nocodazole to induce mitosis. The results, shown in
FIG. 12A and FIG. 13 and table 2 of FIG. 13, illustrated that at
2-10 .mu.M concentrations, most inhibitors specific to Aurora
kinase, Polo like kinase (PLK), Pim1 and Mps1 kinase failed to
affect nocodazole-induced iASPP modification. In contrast, several
inhibitors that affect the activity of cyclin B/cdk1 are able to
prevent iASPP modification to various levels. The most potent
inhibitor of iASPP modification in the presence of nocodazole is
K00616a, an inhibitor that has the lowest IC50 for cyclin B1/cdk1
kinase activity among the panel (9 nM) (FIG. 12A). This finding is
consistent with the fact that Aurora, PLK, Pim1, and Msp1 do not
phosphorylate SP sites, whereas all modified iASPP sites are SP
sites (Johnson 2011). Interestingly, under the same conditions, the
cdk2 inhibitor roscovitine (CYC202/K0020) failed to affect iASPP
modification, supporting the notion that iASPP modification in
mitosis is predominantly caused by cyclin B1/cdk1. Hence, the
LX128.5 negative and slow-migrating iASPP induced in mitosis is
phosphorylated by cyclin B1/cdk1 in vivo and it can be inhibited by
K00616a.
[0377] The ability of K00616a to reactivate wild type p53 function
in melanoma cells was tested in three melanoma cell lines that all
express B-RAFV600E. WM278 and Me300 also express wild type p53,
whereas SK-MEL37 expresses mutant p53. Cells were first treated
with various combinations of Nutlin3 and K00616a in the presence or
absence of RNAi of iASPP and of p53. Wild type p53 function was
measured by a p53-dependent apoptosis assay, detecting the
percentage of cells containing subG1 DNA content and growth
suppression in a clonogenic survival assay. Under the conditions
used, Nutlin3 or K00616a alone resulted in very low rates of
apoptotic cell death. Similarly, their ability to suppress cell
growth was also limited. The activities of Nutlin3 and K00616a were
confirmed by an increase in the expression of p53 as well as the
percentage of G1 or G2/M cells after the treatment (FIG. 13B).
Interestingly, iASPP RNAi resulted in a two-fold increase in
apoptosis, accompanied by a clear reduction in the number of
colonies. This illustrates that iASPP is able to inhibit p53
independently of mdm2. Additionally, a treatment with K00616a plus
Nutlin3a resulted in a two- to three-fold increase in apoptosis in
both Me300 and WM278 cells. This was again accompanied with a
decrease in the number of colonies detected. Importantly, the
apoptosis induced by K00616a and Nutlin3 is iASPP and
p53-dependent. RNAi of iASPP had little effect on K00616a and
Nutlin3 induced apoptosis whereas p53RNAi prevented both the
apoptosis and growth suppression induced by K00616a+Nutlin3.
Consistent with this, K00616a+Nutlin3 had minimal impact in
inducing apoptosis and growth suppression in SK-MEL37, which
expresses mutant p53 (FIG. 12B).
[0378] Since K00616a+Nutlin3 is able to restore wild type p53
function in BRAFV200E expressing melanoma cell lines, WM278 and
Me300, we examined whether restoring wild type p53 function and
concurrently inhibiting B-RAFV600E can cause synthetic lethality.
WM278, Me300 and SK-MEL37 were treated either with PLX4032,
K00616a, Nutlin3 or in combination as labelled. In agreement with
previous reports (Poulikakos et al., 2011), PLX4032 alone is able
to induce apoptosis and growth suppression in all three cell lines
tested regardless of their p53 status. K00616a or Nutlin3 alone had
minimal effect on the growth suppression effects of PLX4032. High
levels of apoptosis and growth suppression were observed in WM278
and Me300 cells, but not in SK-MEL37 cells, when K00616a, Nutlin3
and PLX4032 were used together (FIG. 12C). These data illustrate
that the tumour suppressive function of wild type p53 can be
reactivated in melanoma cells by inhibiting phosphorylated nuclear
iASPP and mdm2. Restoring wild type p53 and inhibiting B-RAFV600E
may cause enhanced killing of melanoma cells and could therefore
provide a new strategy to treat melanomas.
[0379] Discussion:
[0380] Acquired drug resistance is the major challenge for
molecularly targeted cancer therapy. Thus, the identification of
ways to treat cancer cells with combined agents is of great
interests. Here we show that restoring wild type p53 function and
inhibiting B-RAFV600E represents a novel approach to causing
synthetic inhibition in human melanoma cells. Detailed
understanding of the loss of tumour suppressive function by wild
type p53 in human melanoma cells led us to discover that, in
addition to mdm2, p53 is inhibited by phosphorylated nuclear iASPP
in melanoma cells. Knowing that deregulated cyclin B1/cdk1 may
phosphorylate iASPP and cause its nuclear localisation, which
increases its binding and inhibition of p53, led us to identify a
cyclinB/cdk1 inhibitor, K00616a, as a potential agonist of p53.
When used together with Nutlin3, it acted synergistically to
restore wild type p53 function in melanoma cells. We also observed
that activating p53 using K00616a plus Nutlin3 with concurrent
inhibition of BRAFV600E with PLX4032 resulted in the highest
observed levels of apoptosis and growth suppression in melanoma
cells.
[0381] These studies are consistent with our finding that nuclear
iASPP is significantly associated with lymph node metastasis
melanomas in vivo. It is likely that all these observations relate
to a deregulation of cyclin B1/cdk1 and an increase in iASPP Ser84
and Ser113 phosphorylation.
[0382] Besides cyclinB1 and cdk1, we further screened the
expression pattern of other 24 key proteins involved in
tumorgenesis of melanomas. Among these 24 proteins, the interested
ASPP2, MDM2 family members, melanoma-associated antigens, familial
melanomas candidate p16ink4a, Braf-Brn2-MITF-TBX2 pathway
components, mitosis related cdks and cyclins, cdk5 and regulators,
and prolyl hydroxylases do not show significant correlation with
iASPP modify pattern, except for p53 and MAGE-C2. While MAGE-C2
show relatively weak correlation with iASPP modify pattern, p53
show strong correlation with iASPP modify pattern in both 18
melanomas and 7 prostate cancer cells. p53 cDNA were thus amplified
from melanomas and their statuses were sequenced. As a result cells
with a low ratio of modified iASPP universally host mutated p53
(6/7 for melanomas, 5/5 for prostate. Me248.3 is the only
exception; it hosts wild type p53 mRNA, but exhibits null p53
protein level for an unknown reason. Cells with high ratio of
modified iASPP universally host wt p53 (10/11 for melanomas, 2/2
for prostate). IGR37, the only exception with mutated p53 but a
high iASPP modification ratio, is derived from a same patient with
primary IGR39. IGR37 is established to be a malignant melanoma cell
line, described to be very metastatic and tumorigenic in nude model
and confirmed in human. It show high iASPP modify ratio comparing
with primary IGR39 (.about.80% vs .about.15%). It implied that
iASPP might also be preferentially modified in metastatic cells.
This implication was confirmed in prostate LNCaP3, the cell line
derived from LNCaP after 3 times metastasis in mouse. Consistent
with IGR37/39 couple, LNCaP3 show significantly increased iASPP
modify ratio comparing with LNCaP (FIG. 6d, .about.75% vs
.about.35%). The trend was also partially fit in head and neck cell
couples, though it is not as dramatic as in melanomas or prostate
(FIG. 6d).
[0383] Metastatic melanoma has historically been one of the most
treatment refractory types of cancer due to its resistance to
apoptosis (Soengas Lowe 2003) and high metastatic potential
(Flaherty, 2011). Mutation of p53 is a common feature of cancer,
and there is emerging evidence that mutant p53 promotes cancer
metastasis, but a large percentage of melanomas are highly
metastatic whie expressing wild type p53. It is currently estimated
that around 80% of melanomas express wild type p53 and around 50%
are highly metastatic. In an attempt to understand why wild type
p53 loses its tumour suppressive function in metastatic melanoma,
Avery-Kiejda et al. identified abnormal expression of a large
number of p53 downstream target genes, including a failure to
upregulate p53 induced pro-apoptotic genes such as Bax and PIG3 in
melanomas (Avery-Kiejda et al., 2011). Similarly, Houben et al.
also reported defects in the transcriptional activity of p53 using
reporters of p53 target genes (Houben et al., 2011). All these
studies agree with our finding here that the transcriptional
activity, and in particularly the apoptotic function, of p53 is
largely inactivated in melanomas. The tight association between the
expression levels of phosphorylated nuclear iASPP and wild type p53
status (over 90%) supports the notion that the identified
phosphorylated nuclear iASPP represents a key inhibitor of p53 in
melanomas, in addition to mdm2, mdm4 and other cellular inhibitors
(Polsky et al., 2002; Schittek et al., 2007). This is supported by
the finding that the tumour suppressive function of p53 in
melanomas can only be restored by inhibiting both mdm2 and iASPP
phosphorylation, using Nutlin3 and K00616a, respectively.
[0384] The results demonstrated herein is a proof of principle that
targeting two independent pathways that are important in melanoma
development and maintenance may prove to be fertile ground for the
development of strategies to treat malignant melanoma. Furthermore,
the identified mechanism by which cyclin B1/cdk1 phosphorylated
nuclear iASPP is a potent inhibitor of p53 suggests that nuclear
iASPP may be a good biomarker for the tumour suppressive function
of p53 in tumours that express wild type p53.
Example 9
Phosphorylation of iASPP at S84/S113 by Cyclin B1/Cdk1 Contributes
to iASPP Slow Migration and Nuclear Localization in Multiple
Melanoma Cell Lines
[0385] It has been shown in human NSCLC H1299 cells that the
phosphorylation of iASPP at Ser84/Ser113 by cyclin B1/cdk1
contributes to iASPP slow migration (see Example 4). Similar
experiments were performed to assess iASPP phophorylation in other
cell lines. In addition to those reported observations in human
NSCLC H1299 cells, the phosphorylation of iASPP at S84/S113 by
cyclin B1/cdk1 contributes to iASPP slow migration and nuclear
localization in SK-MEL37, B16, and WM278 melanoma cells. These
experimental results confirm that the observed regulation of iASPP
migration is a general phenomenon in melanoma cells.
Example 10
JNJ-7706621, an Inhibitor of Cyclin B1/CDK1, Nocodazole-Induced
iASPP Modification
[0386] Nocodazole was used to induce cells to enter into mitosis, a
cellular state in which CDK kinases are active. To determine if
other kinase or CDK-specific inhibitors could inhibit
nocodazole-induced iASPP modification in H1299 cells, experiments
were carried out with a panel of 15 G2/M kinase inhibitors.
[0387] Immunohistochemistry Staining and Patient Survival
Analysis.
[0388] Immunohistochemical staining was similar to that described
previously (Notari et al., Proc. Natl. Acad. Sci. USA 108,
16645-50, 2011). Tissue sections were dewaxed, rehydrated and
incubated with 3% hydrogen peroxide in methanol to block endogenous
peroxidase activity (10 min). Prior to incubation, antigen
retrieval was carried out in microwaved sodium citrate buffer (pH
6.0) for 10 min for cyclin B1 staining only. Sections were then
blocked with normal goat serum (NGS), incubated overnight at
4.degree. C. with the primary antibody, followed by the
biotinylated secondary antibody for 40 min at room temperature.
Avidin-Biotin Immunoperoxidase-Complex Vectasatin Elite Reagent was
used to amplify the signal and the peroxidase substrate Vector VIP
was used to visualize the complexes. Sections were counterstained
with Methyl Green, dehydrated, permanently mounted, and
photographed. Expression of iASPP in the nucleus was scored as the
product of staining intensity (0-none, 1-weak, 2-moderate,
3-strong) and the proportion of cells exhibiting this stain (0-100%
in 5-10% increments) to generate an Overall Expression score
(0-300). All tumor cells showed cyclin B1 expression. Therefore,
expression levels were compared using an intensity score only
(1-weak, 2-moderate, 3-strong). Statistical analysis was undertaken
in SPSS. Difference between groups were analyzed by the
Mann-Whitney test (U) where the effect size is denoted by r=z/ n
(>0.3=medium effect, >0.5=large effect). The relationship
between nuclear iASPP, dichotomized around the upper quartile, in
primary and metastatic tumors was evaluated using a Chi-squared
test where the effect size was determined by the odds ratio. For
survival analysis, a multivariate Cox proportional hazards model
was used, controlling for known prognostic factors (including age,
gender, site, thickness, and ulceration).
[0389] Xenograft Assay.
[0390] At day 1, C57BL/6 mice (7-9 weeks old) were injected s.c. at
flanks with 1.times.10.sup.6 B16 melanoma cells in 100 .mu.l PBS
(component treated cells for FIG. 19C, untreated cells for FIGS.
19D and 20D). Component (Nutlin3 (40 mg/kg) or JNJ-7706621 (also
referred to herein as "JNJ", 30 mg/kg)) was dissolved in DMSO and
i.p. administered every two days since day 3, or day 7 (no
component administration for FIG. 19C). Tumor size was measured
every two days with vernier caliper. Tumor volumes were calculated
using the following formula: (L*W*W)/2, in which L represents the
larger diameter of the tumor, and W represents the smaller
diameter. On day 13 (tumor area reach .about.1.2 cm diameter in
DMSO group), all mice were sacrificed and tumors were isolated and
weighed.
[0391] FACS analysis and colony formation assay. Cells were
incubated with either DMSO or the cyclin B1/cdk1 inhibitor
JNJ-7706621 (0.4 .mu.M) for 48 hours to block iASPP modification.
Cells were digested and transfected with siRNA using DharmaFECT 1
Transfection Reagent kit (T-2001-02) and seeded on 6-well plates in
fresh medium containing DMSO, JNJ (0.4 .mu.M), Nutlin3 (10-20
.mu.M), PLX4032 (1-5 .mu.M), or indicated combinations for 72
hours. Treated cells were collected and used for the following
three assays. For Sub-G1 analysis, cells were fixed in ice-cold 70%
EtOH. After washing, cells were incubated in PBS containing 0.05
mg/ml RNase A and 0.05 mg/ml propidium iodide (P4864, Sigma) for 30
minutes at room temperature. The percentage of subG1 DNA
content-containing cells was determined by FACS analysis using BD
FACSCanto Flow Cytometer (BD Biosciences) and was used as
indication of apoptosis. For Annexin V/7-AAD analysis, 100 .mu.l
PBS washed cells (1.times.10.sup.6 cells/ml) were incubated in
binding buffer (BD Pharmingen, 559763) with 5 .mu.l Annexin V and 5
.mu.l 7-AAD for 15 min at room temperature in the dark. Binding
buffer (400 .mu.l) was added and cells were subjected to FACS
analysis for 1 hour. The percentage of Annexin V positive cells was
used as indication of apoptosis. For colony formation assays, 100,
1000 or 10,000 cells/well were seeded in 6-well plates and kept in
culture for 8-14 days. Fresh medium was replaced every three
days.
[0392] S84/S113 Phosphorylated Nuclear iASPP is Enriched in Wild
Type p53 Expressing Melanoma Cells.
[0393] As demonstrated herein, melanoma cell lines have deregulated
cyclin B1 in cells in interphase, which is associated with iASPP
modification and nuclear localization. The association between
cyclin B1 and nuclear iASPP expression was examined in human
melanomas. Consistent with the in vitro finding, a strong
correlation between strong cyclinB1 staining and high nuclear iASPP
expression was observed in human melanomas (FIG. 18A), which is
associated with poorer patient survival.
[0394] To investigate the relationship between cyclin B1 and
nuclear iASPP in primary tumors and in metastasis, samples were
divided into six groups depending on how they expressed iASPP and
cyclin B (low or high iASPP with either low, medium, or high cyclin
B (FIG. 18B)). Tumors that showed weak or low cyclinB1 and low
nuclear iASPP expression were shown to be predominantly primary
melanomas. Tumors that showed strong or high cyclin B1
staining/expression and high nuclear iASPP expression were mainly
found in metastasis (FIG. 18B, bar graph). In a cohort of 66
melanoma patients, high nuclear iASPP was significantly associated
with poor survival, whereas strong cyclinB1/low nuclear iASPP was
not associated with poor survival. The data therefore indicates
that deregulated cyclin B1 alone may not be sufficient to affect
survival (FIG. 18C, survival curve). Without being bound by theory,
these results suggest that deregulated cyclin B1/cdk1 may
phosphorylate iASPP in interphase and enhance its ability to
inhibit p53.
[0395] Restoring p53 by Inhibiting Mdm2 and iASPP Suppresses
Melanoma Growth In Vitro and In Vivo.
[0396] Knocking down cyclin B1 with RNAi abolished the
slow-migrating iASPP in the presence of nocodazole. Increased
expression of cyclin B1, but not cdk1, induced nuclear localization
of iASPP in H1299 cells, confirming that cdk1 is present in excess
in cells and cyclin B1 dictates the kinase activity of cyclin
B1/cdk1 and cyclin B1/cdk1 phosphorylates iASPP and causes its
nuclear entry (FIG. 19A).
[0397] A panel of 15 G2/M kinase inhibitors was tested for their
ability to prevent nocodazole induced iASPP modification in H1299
cells. At 2-10 .mu.M concentrations, most inhibitors against Aurora
kinase, Polo-like kinase (PLK), Pim1, or Mps1 kinase failed to
abrogate nocodazole-induced iASPP modification. In contrast,
several inhibitors that are known to affect the activity of cyclin
B1/cdk1 were able to inhibit iASPP modification. The most potent
inhibitor of iASPP modification in the presence of nocodazole was
JNJ-7706621 (JNJ) (Emanuel et al., Cancer Res. 65(19):9038-46,
2005, incorporated herein by reference in its entirety), a cdk1
inhibitor with an in vitro IC50 of 9 nM for cyclin B/cdk1. Under
the same conditions, the cdk2 inhibitor Roscovitine only weakly
affected iASPP modification (FIG. 19A and FIG. 20A: Table 2). To
provide supporting evidence that p53 selectively binds
cyclinB1/cdk1 phosphorylated nuclear iASPP, WM278 cells were
treated with DMSO (control), nocodazole, or JNJ. The treated cell
lysates were incubated in the presence or absence of a Ser/Thr
phosphatase (calf intestine phosphatase, CIP) for 1 hour before
immunoprecipitation. JNJ alone reduced the amount of modified iASPP
whereas nocodazole induced iASPP modification. CIP treatment was
able to abolish nocodazole induced iASPP modification. Importantly,
p53 failed to co-immunoprecipitate iASPP in JNJ, control+CIP and
Nocodazole+CIP-treated lysates, even though p53 was efficiently
co-immunoprecipitated with modified iASPP in both control and
nocodazole treated WM278 cells (FIG. 20A). These data illustrate
that p53 selectively binds phosphorylated iASPP in vivo.
[0398] JNJ demonstrated activity similar to activity of K00616a.
For example, JNJ was able to prevent iASPP from binding to p53.
Knowing that JNJ blocks iASPP from binding to p53, the ability of
JNJ to reactivate wild type p53 function in melanoma cells was
tested in four melanoma cell lines: B16, WM278, Me300, and
SK-MEL37. B16, WM278, and Me300 express wild type p53, whereas
SK-MEL37 expresses mutant p53. Cells were first treated with
various concentrations of Nutlin3 to prevent degradation of p53.
Optimal concentrations of Nutlin3 to induce maximal p53 expression
were identified as 2 .mu.M for SK-MEL37, 10 .mu.M for B16, and 20
.mu.M for both WM278 and Me300 cells (FIG. 20B). Cells were then
treated with various combinations of Nutlin3 and JNJ in the
presence or absence of RNAi for iASPP and for p53. Nutlin3 or JNJ
alone induced apoptosis in only a small percentage of cells. The
ability of Nutlin3 or JNJ to suppress cell growth was also limited,
even though both Nutlin3 and JNJ were able to induce p53 expression
or G2/M arrest, respectively. In B16, Me300, and WM278 cells,
treatment with iASPP RNAi or JNJ resulted in a 3-4 fold increase in
Nutlin3-induced apoptosis, which was determined by the presence of
cells expressing Annexin V (FIG. 19B) or containing Sub-G1 DNA
content (FIGS. 20B and 20C). Apoptosis induced by JNJ+Nutlin3 was
iASPP- and p53-dependent. In the presence of iASPP RNAi, JNJ failed
to further enhance Nutlin3 induced apoptosis. p53 RNAi largely
prevented both apoptosis and growth suppression induced by
JNJ+Nutlin3. JNJ+Nutlin3 had minimal impact in inducing apoptosis
and growth suppression in SK-MEL37 melanoma cells (FIG. 20C).
[0399] Mouse B16 melanoma cells are known to form highly aggressive
melanoma in vivo in immune competent C57BL/6 syngeneic mice.
Because regulation of p53 by iASPP and mdm2 exists in both human
WM278 cells and mouse B16 cells, experiments were carried out to
determine if restoring p53 function in B16 cells is able to
suppress tumor growth in C57BL/6 mice in vivo. B16 cells, treated
with (1) DMSO, (2) Nutlin3, (3) JNJ, or (4) Nutlin3+JNJ, were
injected into the flank of 7-9 week-old female C57BL/6 mice. At 13
days after injection, tumor size and weight in mice injected with
JNJ, Nutlin3 or JNJ+Nutlin3-treated B16 cells was around 40%, 60%
or 15%, respectively, of control (tumor size and weight in
DMSO-treated B16 cell group (FIG. 19C). JNJ was a more potent
inhibitor of B16 growth in vivo than in the colony assay.
[0400] The ability of Nutlin3+JNJ to inhibit B16 melanoma growth in
vivo was further tested by inoculating untreated B16 cells into
C57BL/6 mice three or seven days prior to the intraperitoneal
administration of DMSO, Nutlin3, JNJ or Nutlin3+JNJ, every two days
as indicated. Treatment with Nutlin3+JNJ together was able to
suppress tumor weight/size of B16 melanomas by 61-66% compared to
that of control (DMSO). Administration of Nutlin3 at day 3 or day 7
after B16 inoculation suppressed B16 melanoma growth by 22% or 5%,
respectively. JNJ treatment at both time points suppressed B16
melanoma growth by 36% or 30%, respectively in vivo (FIGS. 19D and
20D). Without being bound by theory, these results indicate that
the timing to inhibit mdm2 in vivo may affect the efficacy of
Nutlin3. However, the efficacy of JNJ or JNJ+Nutlin3 in inhibiting
B16 melanoma growth was less sensitive to administration time.
[0401] Both cytoplasmic and nuclear iASPP were detected in
DMSO-treated B16 tumors, and under the same conditions, p53
expression was almost undetectable. Treatment with JNJ clearly
reduced nuclear iASPP expression in B16 tumors, while it had no
effect on p53 expression. Nutlin3 treatment induced nuclear p53
expression and had minimal impact on iASPP expression. Treatment
with JNJ+Nutlin3 resulted in a reduction in nuclear iASPP and an
increase in nuclear p53 (FIG. 19E). Thus, it was found that the
tumor-suppressive effect of JNJ is associated with a reduction in
nuclear iASPP expression in B16 tumor cells in vivo. Combined
treatment with JNJ and Nutlin3 is able to restore the tumor
suppressor function of p53 and to inhibit melanoma growth in
vivo.
[0402] Restoring the Apoptotic Function of p53 Cooperates with
BRAFV600E Inhibition to Suppress Human Melanoma Cell Growth.
[0403] WM278, Me300 and SK-MEL37 express BRAFV600E, whereas B16
express wild type BRAF. Experiments were carried out to examine
whether restoring wild type p53 function and concurrently
inhibiting BRAFV600E shows cooperative effects. WM278, Me300, and
SK-MEL37 cells were first treated with increasing amounts of
PLX4032. One .mu.M PLX4032 was identified as the optimal
concentration to induce apoptosis in these cells (FIG. 22A).
PLX4032 alone was able to induce apoptosis and growth suppression
in WM278, Me300 and SK-MEL37 cells, regardless of their p53 status,
but had minimal effect in B16 cells (FIG. 21A, FIGS. 22B and C).
Highest levels of apoptosis and growth suppression were observed in
WM278 and Me300 cells when JNJ, Nutlin3 and PLX4032 were used
together. Restoring the apoptotic function of p53 together with
BRAFV600E inhibition can achieve additive effects in suppressing
melanoma growth. In some aspects, targeting two independent
pathways represents a new strategy for the treatment of
melanoma.
[0404] The invention has been described in terms of particular
embodiments found or proposed to comprise specific modes for the
practice of the invention. Various modifications and variations of
the described invention will be apparent to those skilled in the
art without departing from the scope and spirit of the invention.
Although the invention has been described in connection with
specific embodiments, it should be understood that the invention as
claimed should not be unduly limited to such specific embodiments.
Indeed, various modifications of the described modes for carrying
out the invention that are obvious to those skilled in the relevant
fields are intended to be within the scope of the following
claims.
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Sequence CWU 1
1
1512487DNAHomo sapiens 1atggacagcg aggcattcca gagcgcgcgg gactttctgg
acatgaactt ccagtcgctg 60gccatgaaac acatggatct gaagcagatg gagctggaca
cggcggcggc caaggtggat 120gaactgacca agcagctgga gtcgctgtgg
tcagactctc ccgcgcctcc tggcccgcag 180gccggacccc cttctaggcc
gccccggtac agctccagct cgatccctga gcccttcggc 240agccgagggt
ccccccggaa ggcggccacc gacggcgcag acaccccgtt cggacgatca
300gagagtgccc caaccctaca cccctacagc ccgctgtccc ccaagggacg
gccgtcgtcg 360ccgcgcaccc cgctctacct gcagccggac gcctacggca
gcctggaccg cgcgacctcg 420ccccggcccc gcgccttcga tggcgcaggc
agctccctcg gccgtgcgcc ctccccgcgg 480cccgggccag gcccgctccg
ccagcagggt ccccccacgc ctttcgactt cctgggccgc 540gcaggctccc
cccgcggcag ccccctggcg gaggggcccc aggccttctt ccccgagcgt
600gggccgtcac cgcgcccccc tgccacagcc tacgacgcgc cagcgtccgc
cttcgggagc 660tccctgctag gctccggcgg cagcgcattc gccccgcctc
tgcgcgcgca agacgacctg 720acgctgcgcc ggcggcctcc gaaagcctgg
aacgagtctg acctggacgt ggcgtacgag 780aagaagcctt cgcagacagc
gagctatgaa cgcctggacg tcttcgcaag gcctgcctcg 840ccgagcctgc
agctgttgcc ttggagggag agcagcctgg atggactggg gggcaccggc
900aaggacaacc tcactagcgc caccctgccg cgcaattaca aggtctctcc
tctggccagc 960gaccggcgtt cagacgcggg cagctaccgg cgctcgctgg
gctccgcggg gccgtcgggc 1020actttgcctc gcagctggca gcccgtcagc
cgcatcccca tgcccccctc cagcccccag 1080ccccgcgggg ccccgcgcca
gcgtcccatc cccctcagca tgatcttcaa gctgcagaac 1140gccttctggg
agcacggggc cagccgcgcc atgctccctg ggtcccccct cttcacccga
1200gcacccccgc ctaagctgca gccccaacca caaccacagc cccagccaca
atcacaacca 1260cagccccagc tgcccccaca gccccagacc caaccccaaa
cccctacccc agccccccag 1320catccccaac agacatggcc ccctgtgaac
gaaggacccc ccaaaccccc caccgagctg 1380gagcctgagc cggagataga
ggggctgctg acaccagtgc tggaggctgg cgatgtggat 1440gaaggccctg
tagcaaggcc tctcagcccc acgaggctgc agccagcact gccaccggag
1500gcacagtcgg tgcccgagct ggaggaggtg gcacgggtgt tggcggaaat
tccccggccc 1560ctcaaacgca ggggctccat ggagcaggcc cctgctgtgg
ccctgccccc tacccacaag 1620aaacagtacc agcagatcat cagccgcctc
ttccatcgtc atggggggcc agggcccggg 1680gggccggagc cagagctgtc
ccccatcact gagggatctg aggccagggc agggccccct 1740gctcctgccc
caccagctcc cattccaccc ccggccccgt cccagagcag cccaccagag
1800cagccgcaga gcatggagat gcgctctgtg ctgcggaagg cgggctcccc
gcgcaaggcc 1860cgccgcgcgc gcctcaaccc tctggtgctc ctcctggacg
cggcgctgac cggggagctg 1920gaggtggtgc agcaggcggt gaaggagatg
aacgacccga gccagcccaa cgaggagggc 1980atcactgcct tgcacaacgc
catctgcggc gccaactact ctatcgtgga tttcctcatc 2040accgcgggtg
ccaatgtcaa ctcccccgac agccacggct ggacaccctt gcactgcgcg
2100gcgtcgtgca acgacacagt catctgcatg gcgctggtgc agcacggcgc
tgcaatcttc 2160gccaccacgc tcagcgacgg cgccaccgcc ttcgagaagt
gcgaccctta ccgcgagggt 2220tatgctgact gcgccaccta cctggcagac
gtcgagcaga gtatggggct gatgaacagc 2280ggggcagtgt acgctctctg
ggactacagc gccgagttcg gggacgagct gtccttccgc 2340gagggcgagt
cggtcaccgt gctgcggagg gacgggccgg aggagaccga ctggtggtgg
2400gccgcgctgc acggccagga gggctacgtg ccgcggaact acttcgggct
gttccccagg 2460gtgaagcctc aaaggagtaa agtctag 24872828PRTHomo
sapiens 2Met Asp Ser Glu Ala Phe Gln Ser Ala Arg Asp Phe Leu Asp
Met Asn 1 5 10 15 Phe Gln Ser Leu Ala Met Lys His Met Asp Leu Lys
Gln Met Glu Leu 20 25 30 Asp Thr Ala Ala Ala Lys Val Asp Glu Leu
Thr Lys Gln Leu Glu Ser 35 40 45 Leu Trp Ser Asp Ser Pro Ala Pro
Pro Gly Pro Gln Ala Gly Pro Pro 50 55 60 Ser Arg Pro Pro Arg Tyr
Ser Ser Ser Ser Ile Pro Glu Pro Phe Gly 65 70 75 80 Ser Arg Gly Ser
Pro Arg Lys Ala Ala Thr Asp Gly Ala Asp Thr Pro 85 90 95 Phe Gly
Arg Ser Glu Ser Ala Pro Thr Leu His Pro Tyr Ser Pro Leu 100 105 110
Ser Pro Lys Gly Arg Pro Ser Ser Pro Arg Thr Pro Leu Tyr Leu Gln 115
120 125 Pro Asp Ala Tyr Gly Ser Leu Asp Arg Ala Thr Ser Pro Arg Pro
Arg 130 135 140 Ala Phe Asp Gly Ala Gly Ser Ser Leu Gly Arg Ala Pro
Ser Pro Arg 145 150 155 160 Pro Gly Pro Gly Pro Leu Arg Gln Gln Gly
Pro Pro Thr Pro Phe Asp 165 170 175 Phe Leu Gly Arg Ala Gly Ser Pro
Arg Gly Ser Pro Leu Ala Glu Gly 180 185 190 Pro Gln Ala Phe Phe Pro
Glu Arg Gly Pro Ser Pro Arg Pro Pro Ala 195 200 205 Thr Ala Tyr Asp
Ala Pro Ala Ser Ala Phe Gly Ser Ser Leu Leu Gly 210 215 220 Ser Gly
Gly Ser Ala Phe Ala Pro Pro Leu Arg Ala Gln Asp Asp Leu 225 230 235
240 Thr Leu Arg Arg Arg Pro Pro Lys Ala Trp Asn Glu Ser Asp Leu Asp
245 250 255 Val Ala Tyr Glu Lys Lys Pro Ser Gln Thr Ala Ser Tyr Glu
Arg Leu 260 265 270 Asp Val Phe Ala Arg Pro Ala Ser Pro Ser Leu Gln
Leu Leu Pro Trp 275 280 285 Arg Glu Ser Ser Leu Asp Gly Leu Gly Gly
Thr Gly Lys Asp Asn Leu 290 295 300 Thr Ser Ala Thr Leu Pro Arg Asn
Tyr Lys Val Ser Pro Leu Ala Ser 305 310 315 320 Asp Arg Arg Ser Asp
Ala Gly Ser Tyr Arg Arg Ser Leu Gly Ser Ala 325 330 335 Gly Pro Ser
Gly Thr Leu Pro Arg Ser Trp Gln Pro Val Ser Arg Ile 340 345 350 Pro
Met Pro Pro Ser Ser Pro Gln Pro Arg Gly Ala Pro Arg Gln Arg 355 360
365 Pro Ile Pro Leu Ser Met Ile Phe Lys Leu Gln Asn Ala Phe Trp Glu
370 375 380 His Gly Ala Ser Arg Ala Met Leu Pro Gly Ser Pro Leu Phe
Thr Arg 385 390 395 400 Ala Pro Pro Pro Lys Leu Gln Pro Gln Pro Gln
Pro Gln Pro Gln Pro 405 410 415 Gln Ser Gln Pro Gln Pro Gln Leu Pro
Pro Gln Pro Gln Thr Gln Pro 420 425 430 Gln Thr Pro Thr Pro Ala Pro
Gln His Pro Gln Gln Thr Trp Pro Pro 435 440 445 Val Asn Glu Gly Pro
Pro Lys Pro Pro Thr Glu Leu Glu Pro Glu Pro 450 455 460 Glu Ile Glu
Gly Leu Leu Thr Pro Val Leu Glu Ala Gly Asp Val Asp 465 470 475 480
Glu Gly Pro Val Ala Arg Pro Leu Ser Pro Thr Arg Leu Gln Pro Ala 485
490 495 Leu Pro Pro Glu Ala Gln Ser Val Pro Glu Leu Glu Glu Val Ala
Arg 500 505 510 Val Leu Ala Glu Ile Pro Arg Pro Leu Lys Arg Arg Gly
Ser Met Glu 515 520 525 Gln Ala Pro Ala Val Ala Leu Pro Pro Thr His
Lys Lys Gln Tyr Gln 530 535 540 Gln Ile Ile Ser Arg Leu Phe His Arg
His Gly Gly Pro Gly Pro Gly 545 550 555 560 Gly Pro Glu Pro Glu Leu
Ser Pro Ile Thr Glu Gly Ser Glu Ala Arg 565 570 575 Ala Gly Pro Pro
Ala Pro Ala Pro Pro Ala Pro Ile Pro Pro Pro Ala 580 585 590 Pro Ser
Gln Ser Ser Pro Pro Glu Gln Pro Gln Ser Met Glu Met Arg 595 600 605
Ser Val Leu Arg Lys Ala Gly Ser Pro Arg Lys Ala Arg Arg Ala Arg 610
615 620 Leu Asn Pro Leu Val Leu Leu Leu Asp Ala Ala Leu Thr Gly Glu
Leu 625 630 635 640 Glu Val Val Gln Gln Ala Val Lys Glu Met Asn Asp
Pro Ser Gln Pro 645 650 655 Asn Glu Glu Gly Ile Thr Ala Leu His Asn
Ala Ile Cys Gly Ala Asn 660 665 670 Tyr Ser Ile Val Asp Phe Leu Ile
Thr Ala Gly Ala Asn Val Asn Ser 675 680 685 Pro Asp Ser His Gly Trp
Thr Pro Leu His Cys Ala Ala Ser Cys Asn 690 695 700 Asp Thr Val Ile
Cys Met Ala Leu Val Gln His Gly Ala Ala Ile Phe 705 710 715 720 Ala
Thr Thr Leu Ser Asp Gly Ala Thr Ala Phe Glu Lys Cys Asp Pro 725 730
735 Tyr Arg Glu Gly Tyr Ala Asp Cys Ala Thr Tyr Leu Ala Asp Val Glu
740 745 750 Gln Ser Met Gly Leu Met Asn Ser Gly Ala Val Tyr Ala Leu
Trp Asp 755 760 765 Tyr Ser Ala Glu Phe Gly Asp Glu Leu Ser Phe Arg
Glu Gly Glu Ser 770 775 780 Val Thr Val Leu Arg Arg Asp Gly Pro Glu
Glu Thr Asp Trp Trp Trp 785 790 795 800 Ala Ala Leu His Gly Gln Glu
Gly Tyr Val Pro Arg Asn Tyr Phe Gly 805 810 815 Leu Phe Pro Arg Val
Lys Pro Gln Arg Ser Lys Val 820 825 318DNAArtificial
SequenceSynthetic primer 3cggggacact ttgcgttc 18420DNAArtificial
SequenceSynthetic primer 4ttctgacgca cacctattgc 20518DNAArtificial
SequenceSynthetic primer 5gctttccacg acggtgag 18620DNAArtificial
SequenceSynthetic primer 6caagggttca aagacccaaa 20712PRTArtificial
SequenceSynthetic peptide 7Gln Glu Thr Phe Ser Asp Leu Trp Lys Xaa
Leu Pro 1 5 10 812PRTArtificial SequenceSynthetic peptide 8Met Pro
Arg Phe Met Asp Tyr Trp Glu Gly Leu Asn 1 5 10 99PRTArtificial
SequenceSynthetic peptide 9Phe Met Asp Tyr Trp Glu Gly Leu Asn 1 5
1012PRTArtificial SequenceSynthetic peptide 10Glu Thr Phe Xaa Asp
Xaa Trp Lys Xaa Leu Xaa Glu 1 5 10 119PRTArtificial
SequenceSynthetic peptide 11Phe Met Xaa Tyr Trp Glu Xaa Leu Asn 1 5
129PRTArtificial SequenceSynthetic peptide 12Phe Met Xaa Xaa Trp
Glu Xaa Leu Asn 1 5 139PRTArtificial SequenceSynthetic peptide
13Phe Met Xaa Xaa Xaa Glu Xaa Leu Asn 1 5 1413PRTArtificial
SequenceSynthetic peptide 14His Pro Tyr Ser Pro Leu Ser Pro Lys Gly
Arg Pro Ser 1 5 10 1515PRTArtificial SequenceSynthetic peptide
15Pro Glu Pro Phe Gly Ser Arg Gly Ser Pro Arg Lys Ala Ala Thr 1 5
10 15
* * * * *